BACKGROUND
the levels of nonesterified fatty acids  in bovine plasma increase during transition periods, such as after calving and during early lactation  <cit> .
during these stages, a negative energy balance is generated, which is mainly characterized by an increased release of nefas from adipose tissues into the plasma  <cit> .
elevated plasma nefa concentrations have recently been related to the incidence of metabolic and infectious bovine diseases, including fatty hepatitis, metabolic ketosis, mastitis, and endometritis, which cause high economic losses to the cattle industry worldwide  <cit> .
therefore, nefas directly affect tissue/organ functions, and even more importantly, indirectly affect the bovine innate immune system, as previously reported  <cit> .
endothelial cells are highly immunoreactive cells that synthesize a broad spectrum of chemokines, cytokines, and adhesion molecules, and therefore play an active part in early proinflammatory reactions  <cit> .
consistent with this, the exposure of primary bovine aortic endothelial cells  to a mixture of nefas for 24 h altered their membrane phospholipid profile, increasing the stearic acid concentration and reducing the polyunsaturated fatty acid concentrations  <cit> .
in the same study,  <dig>  mm nefa increased the expression of the proinflammatory cytokines interleukin  <dig>  and il- <dig>  and the adhesion molecules intercellular adhesion molecule  <dig>  and vascular cell adhesion molecule  <dig>  in baecs  <cit> , which interact with other immunocompetent cells, such as neutrophils.
although the effects of nefas on bovine leukocytes and endothelial cells have been examined previously  <cit> , the exact molecular mechanisms are still poorly understood.
it has long been accepted that the hydrophobic nature of lipids allows them to activate cells after they enter them, e.g., through membrane transport mechanisms  <cit> .
however, a g-protein-coupled free fatty acid receptor known as gpr <dig> or free fatty acid receptor  <dig>  has recently been described  <cit> .
this novel free fatty acid receptor recognizes saturated and unsaturated medium- and long-chain fatty acids, and the human, rat, and mouse isoforms of ffar1/gpr <dig> have been described  <cit> .
recently, the effects of ffar1/gpr <dig> agonists, such as oleic acid  and linoleic acid , have been demonstrated in epithelial mammary cells  <cit>  and neutrophils  <cit> .
consequently, primary bovine tissues are thought to be susceptible to nefa exposure primarily via the corresponding bovine free fatty acid receptor  <dig> .
based on this evidence, we evaluated whether different types of nefas can rapidly modify the intracellular calcium response in primary bovine endothelial cells exposed to single fatty acids and to study in more detail the molecular mechanisms involved in this endothelial activation.
RESULTS
acute treatment with nefas does not affect the viability of primary bovine umbilical vein endothelial cells
cells exposed to 300 μm la, palmitic acid , oa, myristic acid , or stearic acid  showed no significant difference in the propidium iodide signal for 15 min when compared with untreated cells  .
similar results were observed in buvecs exposed to 1 % vehicle  for the same period.
therefore, exposure to 300 μm of each fatty acid did not increase buvec death any more than triton x- <dig> treatment, used as the positive control.
we demonstrated that  <dig>  mm egta, 50 μm bapta-am or each nefa plus egta or bapta did not affect the viability of buvecs .
these results clearly demonstrate that these fatty acid concentrations and exposure time have no toxic effects on buvecs.
nefas increase the intracellular calcium influx in buvecs
the intracellular calcium response in primary bovine endothelial cells was evaluated in buvecs exposed acutely to different nefas for 100 s. the calcium signal increased quickly after stimulation with 300 μm ma, pa, sa, or oa, with similar kinetics, and the intracellular calcium levels reached a new steady state .
in contrast, la caused a slow but constant increase in intracellular calcium .
to identify the roles of intracellular and extracellular calcium, we used the well-known calcium-chelating agents bapta-am and egta.
incubation with bapta-am reduced the slope of calcium increase by more than 50 % in all cases, except for la .
however, this did not affect the area under the curve  of calcium flux .
moreover, the bapta-am treatment significantly reduced the area under the curve  only in cells previously exposed to la .
the latter suggests that the increase in calcium induced by la is dependent on intracellular and extracellular calcium mobilization.
in our experiments, the nefa-mediated increase in calcium was significantly inhibited in the presence of egta , suggesting that the increase in calcium in the whole experiment is mainly depended on calcium influx.fig.
<dig> intracellular calcium increases caused by nefas are dependent on extracellular calcium.
a–e time courses of representative fura- <dig> ratio signals in at least three assays, caused by 300 μm of each nefa in buvec cells.
each nefa was added at 50 s during a 100 s period .
suspended cells were exposed to each nefa in hbss, without or with pretreatment with 50 μm bapta-am or calcium-free hbss/ <dig>  mm egta .
before fatty acid exposure, egta was added at the light grey arrow.
buvecs were also exposed to only to the vehicle control at 50 s .
ethanol  was used as the vehicle for ma and sa.
dmso  was used as the vehicle for pa, oa, and la.
f–j aucs for nefas in hbs buffer , calcium-free buffer with  <dig>  mm egta , or after bapta treatment .
auc of vehicle alone, 1 % ethanol or 1 % dmso .
the data are expressed as the means ± sem of at least three experiments.
*p <  <dig> , **p <  <dig> , ***p <  <dig>
mechanisms of ma and la effects on intracellular calcium release in buvecs
we investigated the potential differential responses of buvecs to ma and la, exemplary for saturated and unsaturated nefas, respectively.
to investigate the mechanism of the calcium increase, we first constructed dose–response curves of the calcium signal after stimulation with ma and la, and obtained half-maximal effective concentrations  of  <dig> and 37 μm, respectively .
we then used concentrations close to the ec <dig> values to assess the mechanisms involved in calcium flux.
upon stimulation with 100 μm ma, buvecs showed a rapid increase in calcium, which decreased until a new steady state was reached after 30 s .
in contrast, stimulation with 30 μm la caused a sustained increase in calcium within the first 100 s , and a steady state was observed after stimulation for 200 s .fig.
<dig> signaling routes of the calcium responses caused by ma and la in buvecs.
a and d dose–response of calcium signaling induced by myristic acid  or linoleic acid .
the area under the curve  calculated for the first 100 s of the fura-2/am time course.
b and e average time courses for the fura- <dig> ratio in buvecs exposed to 100 μm ma in ethanol or 30 μm la in 1 % dmso.
graph inset: whole time course of the fura-2/am ratio after exposure to 100 μm ma before the addition of 132 μm digitonin  .
c and f calcium response in cells triggered by 1 % ethanol or 1 % dmso, 100 μm ma or 30 μm la alone or after preincubation with 100 μm 2-apb, 10 μm gw <dig>  4 μm u <dig> , or 20 μm nifedipine  for 15 min.
cells were incubated for 16 h with 100 ng/ml pertussis toxin  before ma or la supplementation.
the data are expressed as the means ± sem of at least three independent experiments.
*p <  <dig> , **p <  <dig> , ***p <  <dig>
to evaluate whether ffar1/gpr <dig> actively participates in the calcium increase induced by ma or la, we used its antagonist gw <dig>  as previously reported  <cit> .
the response produced by 100 μm ma was inhibited by 59 % by 10 μm gw <dig> .
this was also observed when other saturated fatty acids were used in our in vitro bovine endothelial cell system, and gw <dig> inhibited  <dig> and 63 % of the calcium response induced by 300 μm pa and sa, respectively .
in contrast, the calcium influx response to 30 μm la was not affected by gw <dig> , and a similar response was observed when oa was used as the unsaturated fatty acid .
to assess the role of the g-protein gαi/o, phospholipase c , and the inositol triphosphate  receptors/calcium-operated channels induced by ma and la, we used pertussis toxin   <cit> , u <dig>  <cit> , and 2-apb , respectively.
we demonstrated that preincubation with 100 ng/ml ptx for 16 h did not inhibit the calcium influx induced either by ma or la fatty acids in buvecs, which suggests that gαi/o does not participate in this type of response.
the mobilization of calcium by la was reduced by 4 μm u <dig>  but the effect of ma was not affected by treatment with the plc inhibitor.
2-apb  reduced both the ma- and la-mediated increases in calcium by  <dig> and 23 %, respectively .
there is some evidence that fatty acids can directly and indirectly activate the l-type calcium channels  in hek <dig>  cos- <dig> and rat insulinoma ins-1e cells in vitro  <cit> .
however, the participation of ltccs in the calcium increase induced by ma or la in buvecs has not been evaluated.
therefore, we investigated whether these channels participate in the calcium increase induced by ma or la stimulation.
our results show that the ltcc blocker nifedipine  inhibited the increases in calcium induced by ma and la by  <dig> and 22 %, respectively .
these results suggest that ltccs participate in the increases in calcium caused by saturated and unsaturated fatty acids in buvecs.
ffar1/gpr <dig> expression and activity
to examine ffar1/gpr <dig> protein expression in buvecs, we used western blotting and immunofluorescence assays directed against the conserved epitope of bovine gpr <dig>  as described elsewhere  <cit> .
an immunoblotting analysis of the total protein extracts of buvecs showed a band with an apparent molecular size of 23-kda, lower than expected but similar to that observed in lovo cells, which were used as the positive control, as suggested by the manufacturer of the anti-gpr <dig> antibody .
the same antibody was used for the immunofluorescence assays of buvecs, which detect the internal region of human gpr <dig>  and therefore the intracellular and plasma membrane distribution .
to confirm that the ffar1/gpr <dig> protein is in its active form in buvecs, we also measured the calcium signal separately with two known ffar1/gpr <dig> agonists, tak- <dig>  <cit>  and gw <dig>  <cit> .
tak- <dig> increased the intracellular calcium in a dose-dependent manner , whereas gw <dig> did not change the calcium signal at either low  or high concentrations .fig.
<dig> protein expression and functional detection of ffar1/gpr <dig> in buvec monolayers.
a western blot of bovine ffar1/gpr <dig>  total protein extract from lovo cells was used as the positive control for two buvec isolates.
50 μg of total protein was loaded onto a 6 % polyacrylamide gel.
b ffar1/gpr <dig> immunofluorescence.
left image, nuclear signals of cells incubated with propidium iodide ; inset image: only the secondary antibody was used, as the negative control, bar = 20 μm.
middle image, ffar1/gpr <dig> detected with rabbit anti-gpr <dig> antiserum and an anti-rabbit igg secondary antibody.
right image, nuclear signals and gpr <dig> images are superimposed, bar = 20 nm.
c representative time course of the fura-2/am ratio in buvecs exposed to 1 % dmso or 100 μm tak- <dig> at the time indicated by the arrow.
d cells were exposed to dmso or tak- <dig>  the area under the curve  was calculated during the first 100 s after exposure to the stimulus.
the data are expressed as the means ± sem of at least three independent experiments.
**p <  <dig> , ***p <  <dig>
la increases nitric oxide  production
to assess whether nefas affect no production in the endothelium, we exposed buvecs to ma, sa, pa, oa, or la and evaluated the cells for no production using daf-fm signals.
only la significantly increased the daf-fm fluorescence intensity in buvec monolayers.
as illustrated in fig.
4a, the daf-fm signal increased approximately  <dig> -fold in la-exposed buvecs compared with the signal in cells treated with vehicle .
the increase in no induced by la was reduced by 25 % when the cells were treated with 100 μm l-name, a potent endothelial nitric oxide synthase  inhibitor .
this indicates that la activates enos in primary bovine endothelial cells.
treatment with bapta, egta, or bapta plus egta reduced the increase in no induced by la by  <dig>   <dig>  and 86 %, respectively .
in contrast, neither 4 μm u <dig>  10 μm gw <dig>  100 μm 2-apb, 20 μm nifedipine, nor 100 ng/ml ptx reduced the la-mediated increase in no .fig.
<dig> nefa effects on nitric oxide production in buvecs.
a basal daf fluorescence , and after treatment with  <dig>  % ethanol, 300 μm myristic acid , 300 μm stearic acid ,  <dig>  % dmso , 300 μm palmitic acid , 300 μm oleic acid , or 300 μm linoleic acid  for 5 min at 37 °c.
b daf fluorescent signal after incubation with 300 μm la for 5 min, normalized to the signal without fatty acids.
the cells were incubated with bapta, egta, or bapta + egta before treatment with la.
c effect of l-name and gw <dig> on the la-induced increase in the daf signal.
basal fluorescence.
daf fluorescence is expressed in arbitrary units .
each bar represents the mean ± sem of at least three independent experiments.
*p <  <dig> , **p <  <dig> , ***p <  <dig>
la but not ma increases icam- <dig> and il- <dig> mrna expression
because only la effectively increased enos activity in buvecs, we tested whether la also affects icam- <dig> or il- <dig> expression.
only la induced the expression of icam- <dig> and il- <dig> mrnas in buvecs, and la-induced il- <dig> expression was significantly inhibited by gw <dig>  however, the expression of icam- <dig> was not reduced by treatment with the ffar1/gpr <dig> antagonist .fig.
<dig> nefa effects on icam- <dig> and il- <dig> expression in buvecs.
cells were treated with  <dig>  % ethanol, 300 μm myristic acid ,  <dig>  % dmso, 300 μm oleic acid , or 300 μm linoleic acid  for 3 h and their rnas were then isolated.
some cells were treated with 10 μm gw <dig> or 4 μm u <dig>  for 30 min before the la treatment.
the expression of icam- <dig> and il- <dig> was analyzed with real-time pcr using specific primers.
relative abundance = 2–Δct, where Δct = cycle treshold icam- <dig> or ct il- <dig> minus cycle treshold sdha.
the data are expressed as the means ± sem of three experiments.
*p <  <dig>
discussion
the nefa levels in blood plasma are particularly elevated during the transition period in dairy cattle, when lipids are strongly mobilized from the adipose tissues to the bloodstream, with plasma concentrations increasing from the basal level of  <dig>  mm to  <dig> –2 mm .
until now, the involvement of g-coupled fatty acid receptors, such as ffar1/gpr <dig>  in the nefa-mediated calcium response in primary endothelial cells has been unclear.
this study has demonstrated that single nefas alone, at low concentrations of  <dig>  mm, increase the intracellular calcium response in primary bovine endothelial cells, inducing different molecular pathways involved in the endothelial cell activation process.
in naturally occurring acute systemic coliform mastitis linoleic acid and oleic acid were the most abundant nefa, suggesting that specific fatty acids can be involved in inflammation and innate immunity, and to be relevant as risk factors during the transition period  <cit> .
different studies have reported the potency with which ma and la bind to human ffar1/gpr <dig> in transfected cho and hek <dig> cells by measuring the calcium signal.
for ma, a ec <dig> value has been described in these cells, close to 7 μm, but for la, the ec <dig> values ranged from  <dig>  to  <dig>  μm  <cit> .
in contrast, the ec <dig> values for ma and la were  <dig>  and  <dig>  μm, respectively, in a reporter cell line based on hela cells transfected with hgpr <dig>  <cit> .
these results are quite similar to those observed in the present study, especially regarding the stimulatory potency of la.
because many of the effects of nefas on primary bovine endothelial cells could be attributed to bgpr <dig>  including increased intracellular calcium influx, the presence of this specific nefa receptor was assessed in buvecs in this study.
we detected ffar1/gpr <dig> expression in buvecs, and also evaluated its effect on the calcium flux using the agonist tak- <dig> in our in vitro primary bovine endothelial cell system.
there is some evidence that ffar1/gpr <dig> is expressed in bovine cells and bovine ffar1/gpr <dig> has been shown to share 84 % nucleotide sequence identity with the human isoform  <cit> .
previous studies have examined the expression of gpr <dig> in endothelial cells from monkey brain tissues  <cit> , neuromicrovascular cells from rodent tissues  <cit> , and human cardiac endothelial cells  <cit> .
to the best of our knowledge, this work is the first to report the expression of gpr <dig> in primary bovine endothelial cells.
our data also suggest that the ma-mediated increase in calcium occurs when ma binds to bgpr <dig>  because it was clearly inhibited by gw <dig>  a potent ffar1/gpr <dig> antagonist.
in contrast, the increase in calcium induced by la does not involve this receptor, according to our antagonist experiments.
we also observed that ip <dig> receptors/calcium-operated channels and ltccs seem to participate in the cellular responses to ma and la, respectively.
others have shown, that oa increased the calcium signal in hek  <dig> cells transiently transfected with the α <dig> subunits of the ltccs  <cit> .
the pathway of calcium entry via the ltccs induced by nefas is evidently cell-type dependent.
ltccs were also associated with the increase in calcium mediated by ffar1/gpr <dig> activation that was induced by the saturated fatty acid pa in rat β-cell line ins-1e  <cit> , by the unsaturated fatty acid oa in islet β-cells from rats  <cit> , and by la in primary cultured chicken hepatocytes  <cit> .
our examination of the cellular calcium flux suggests that ma, but not la, recognizes ffar1/gpr <dig> in buvecs.
the cacna2d <dig> gene encodes the alpha 2/delta subunit which is an accessory part of the voltage-gated calcium channel family  and considered to be an important candidate protein influencing bovine mastitis  <cit> .
although many publications have reported ltcc activity in bovine immune cells, there is not yet any evidence that these channels are expressed in bovine endothelial cells.
in the present study, nifedipine affected buvecs, suggesting the participation of ltccs.
it has also been recently demonstrated that the increase in calcium caused by endothelin- <dig> is ltcc-dependent in cultured bovine corneal endothelial cells  <cit> .
we also evaluated the no response in nefa-exposed bovine endothelium, because no is a well-known indicator of inflammation  <cit> .
all nefas were evaluated, but only la increased no in the endothelial cells.
this response was dependent on both calcium and enos.
there is evidence that enos activity is dependent on ca2+/calmodulin  <cit> , and it is probable that the increase in no is a secondary effect after the interaction of enos with ca+2/calmodulin.
both no and enos actively participate in the regulation of vascular tone and are therefore important intracellular enzymes, especially in microvascular hyperpermeability  <cit> .
there is some evidence that increased levels of no in endothelial cells produce proinflammatory profiles, which might lead to metabolic diseases.
la induces reactive oxygen species  and the overproduction of no after an increase in calcium, causing peroxynitration in endothelial cells  <cit> .
nf-κb, a crucial transcription factor, is then activated by peroxynitrite, a potent oxidative agent, inducing the expression of adhesion molecules  <cit>  and chemokines, such as il- <dig>  <cit> .
we also demonstrated that la, but not ma, induces icam-1- and il-8-expression, suggesting an active role for no in this response.
in bovine endothelial cell cultures, high concentrations of total nefas might increase icam- <dig> mrna and protein expression  <cit> .
the la-induced adhesion molecules and chemokines may participate in leukocyte-endothelial cell interactions, facilitating transendothelial migration  <cit> , and may play a role in the onset of a proinflammatory process during the transition period in dairy cattle.
massive lipid mobilization leading to an increase in plasma nefas is observed in cows around parturition, causing an increased incidence of metabolic and infectious diseases, including laminitis, endometritis, and mastitis .
the exposure of bovine endothelial cells to nefas increases the expression of the proinflammatory cytokines il- <dig> and il- <dig> and the adhesion molecules icam- <dig> and vcam- <dig>  <cit> .
it has also been postulated that the excessive accumulation of nefas during chronic inflammatory responses might increase tnfα, il- <dig>  and il-1β expression by activating nf-κb in adipocytes and macrophages.
these cytokines are thought to link the metabolic state and the inflammatory response, usually in the postpartum period within the uteri of cows  <cit> .
there is evidence that endothelial calcium participates in the transendothelial lymphocyte migration caused by icam- <dig>  <cit> .
moreover, an intracellular calcium increase has been related with icam- <dig> expression in huvec  <cit> .
however, in buvecs, intracellular calcium increase could be not directly associated with the expression of icam- <dig> or il- <dig>  consistently with these assumption ma increased calcium flux was not sufficient to increase the expression of these molecules and the inhibition of plc did not reduce the expression of icam- <dig> and il- <dig> induced by la.
our data also demonstrate that the individual nefas have different effects on endothelial cell activity and diverse mechanisms of action.
la induces the expression of icam- <dig> independently of ffar1/gpr <dig> or plc activity, although the expression of il- <dig> is associated with the activation of ffar1/gpr <dig> in buvecs.
in contrast, our results showed that no was induced by la, in a rather g-protein-coupled-receptor-independent process.
there is some evidence that these receptors need not bind to a g-protein in the presence of high agonist concentrations, which thereby alternatively activate the erk/mapk pathway  <cit> .
it has been suggested that la activates the phosphoinositide 3-kinase, erk1/ <dig>  and p- <dig> mapk pathways in vascular endothelial cells  <cit> .
in our laboratory, we have recently shown that the increase in no caused by la is p38-dependent .
for this reason, it is possible that la activates ffar1/gpr <dig> or another receptor in a g-protein-independent manner.
for instance, we cannot exclude the possibility that the gpr <dig> receptor recognizes la by another mechanism  <cit> , or that la is recognized by gpr <dig>  a medium-chain fatty acid receptor  <cit> , or cd <dig>  a fatty acid transporter protein  <cit> .
only cd <dig> expression has been demonstrated in endothelial cells  <cit> .
it has also been proposed that la mobilizes intracellular calcium via an la metabolite that induces intracellular mitochondrial calcium fluxes  <cit> .
CONCLUSIONS
we have demonstrated for the first time in primary bovine endothelial cells that gpr <dig> is involved in the increase in calcium induced by ma, and conversely, that the la calcium flux is not associated with this receptor.
we have also demonstrated the participation of ip <dig> receptors/calcium-operated channels and ltccs in the cellular responses to ma and la.
we observed ffar1/gpr <dig> expression in the bovine endothelium and confirmed its activity using its agonist, tak- <dig>  only la increased no levels, and this was independent of ffar1/gpr <dig>  only la induced the expression of icam- <dig> and il- <dig>  and this phenomenon is a ffar1/gpr40- dependent cellular process.
these results indicate that the responses of endothelial cells to individual nefas are highly variable.
recently, it has been demonstrated that there is an association of la enhancement in plasma and milk in naturally occurring acute systemic coliform mastitis  <cit> .
therefore, the increase in total nefas that is described as risk factor for the onset of proinflammatory process that occur during transition periods in cattle, should be revised carefully in the future.
further studies are required to determine whether other individual nefas participate in the bovine proinflammatory responses in vivo during the transition period and in other nefa-dependent disorders in cattle.

 the levels of nonesterified fatty acids in bovine plasma increase during transition periods, such as after calving and during early lactation. during these stages, a negative energy balance is generated, which is mainly characterized by an increased release of nefas from adipose tissues into the plasma. elevated plasma nefa concentrations have recently been related to the incidence of metabolic and infectious bovine diseases, including fatty hepatitis, metabolic ketosis, mastitis, and endometritis, which cause high economic losses to the cattle industry worldwide. therefore, nefas directly affect tissue/organ functions, and even more importantly, indirectly affect the bovine innate immune system, as previously reported. endothelial cells are highly immunoreactive cells that synthesize a broad spectrum of chemokines, cytokines, and adhesion molecules, and therefore play an active part in early proinflammatory reactions. consistent with this, the exposure of primary bovine aortic endothelial cells to a mixture of nefas for 24 h altered their membrane phospholipid profile, increasing the stearic acid concentration and reducing the polyunsaturated fatty acid concentrations. in the same study,  mm nefa increased the expression of the proinflammatory cytokines interleukin and il- and the adhesion molecules intercellular adhesion molecule and vascular cell adhesion molecule in baecs, which interact with other immunocompetent cells, such as neutrophils. although the effects of nefas on bovine leukocytes and endothelial cells have been examined previously, the exact molecular mechanisms are still poorly understood. it has long been accepted that the hydrophobic nature of lipids allows them to activate cells after they enter them, e.g., through membrane transport mechanisms. however, a g-protein-coupled free fatty acid receptor known as gpr or free fatty acid receptor has recently been described. this novel free fatty acid receptor recognizes saturated and unsaturated medium- and long-chain fatty acids, and the human, rat, and mouse isoforms of ffar1/gpr have been described. recently, the effects of ffar1/gpr agonists, such as oleic acid and linoleic acid, have been demonstrated in epithelial mammary cells and neutrophils. consequently, primary bovine tissues are thought to be susceptible to nefa exposure primarily via the corresponding bovine free fatty acid receptor. based on this evidence, we evaluated whether different types of nefas can rapidly modify the intracellular calcium response in primary bovine endothelial cells exposed to single fatty acids and to study in more detail the molecular mechanisms involved in this endothelial activation.  acute treatment with nefas does not affect the viability of primary bovine umbilical vein endothelial cells cells exposed to 300 μm la, palmitic acid, oa, myristic acid, or stearic acid showed no significant difference in the propidium iodide signal for 15 min when compared with untreated cells. similar results were observed in buvecs exposed to 1 % vehicle for the same period. therefore, exposure to 300 μm of each fatty acid did not increase buvec death any more than triton x- treatment, used as the positive control. we demonstrated that  mm egta, 50 μm bapta-am or each nefa plus egta or bapta did not affect the viability of buvecs. these results clearly demonstrate that these fatty acid concentrations and exposure time have no toxic effects on buvecs. nefas increase the intracellular calcium influx in buvecs the intracellular calcium response in primary bovine endothelial cells was evaluated in buvecs exposed acutely to different nefas for 100 s. the calcium signal increased quickly after stimulation with 300 μm ma, pa, sa, or oa, with similar kinetics, and the intracellular calcium levels reached a new steady state. in contrast, la caused a slow but constant increase in intracellular calcium. to identify the roles of intracellular and extracellular calcium, we used the well-known calcium-chelating agents bapta-am and egta. incubation with bapta-am reduced the slope of calcium increase by more than 50 % in all cases, except for la. however, this did not affect the area under the curve of calcium flux. moreover, the bapta-am treatment significantly reduced the area under the curve only in cells previously exposed to la. the latter suggests that the increase in calcium induced by la is dependent on intracellular and extracellular calcium mobilization. in our experiments, the nefa-mediated increase in calcium was significantly inhibited in the presence of egta, suggesting that the increase in calcium in the whole experiment is mainly depended on calcium influx.fig. intracellular calcium increases caused by nefas are dependent on extracellular calcium. a–e time courses of representative fura- ratio signals in at least three assays, caused by 300 μm of each nefa in buvec cells. each nefa was added at 50 s during a 100 s period. suspended cells were exposed to each nefa in hbss, without or with pretreatment with 50 μm bapta-am or calcium-free hbss/  mm egta. before fatty acid exposure, egta was added at the light grey arrow. buvecs were also exposed to only to the vehicle control at 50 s. ethanol was used as the vehicle for ma and sa. dmso was used as the vehicle for pa, oa, and la. f–j aucs for nefas in hbs buffer, calcium-free buffer with  mm egta, or after bapta treatment. auc of vehicle alone, 1 % ethanol or 1 % dmso. the data are expressed as the means ± sem of at least three experiments. *p < , **p < , ***p <  mechanisms of ma and la effects on intracellular calcium release in buvecs we investigated the potential differential responses of buvecs to ma and la, exemplary for saturated and unsaturated nefas, respectively. to investigate the mechanism of the calcium increase, we first constructed dose–response curves of the calcium signal after stimulation with ma and la, and obtained half-maximal effective concentrations of and 37 μm, respectively. we then used concentrations close to the ec values to assess the mechanisms involved in calcium flux. upon stimulation with 100 μm ma, buvecs showed a rapid increase in calcium, which decreased until a new steady state was reached after 30 s. in contrast, stimulation with 30 μm la caused a sustained increase in calcium within the first 100 s, and a steady state was observed after stimulation for 200 s.fig. signaling routes of the calcium responses caused by ma and la in buvecs. a and d dose–response of calcium signaling induced by myristic acid or linoleic acid. the area under the curve calculated for the first 100 s of the fura-2/am time course. b and e average time courses for the fura- ratio in buvecs exposed to 100 μm ma in ethanol or 30 μm la in 1 % dmso. graph inset: whole time course of the fura-2/am ratio after exposure to 100 μm ma before the addition of 132 μm digitonin. c and f calcium response in cells triggered by 1 % ethanol or 1 % dmso, 100 μm ma or 30 μm la alone or after preincubation with 100 μm 2-apb, 10 μm gw 4 μm u, or 20 μm nifedipine for 15 min. cells were incubated for 16 h with 100 ng/ml pertussis toxin before ma or la supplementation. the data are expressed as the means ± sem of at least three independent experiments. *p < , **p < , ***p <  to evaluate whether ffar1/gpr actively participates in the calcium increase induced by ma or la, we used its antagonist gw as previously reported. the response produced by 100 μm ma was inhibited by 59 % by 10 μm gw. this was also observed when other saturated fatty acids were used in our in vitro bovine endothelial cell system, and gw inhibited and 63 % of the calcium response induced by 300 μm pa and sa, respectively. in contrast, the calcium influx response to 30 μm la was not affected by gw, and a similar response was observed when oa was used as the unsaturated fatty acid. to assess the role of the g-protein gαi/o, phospholipase c, and the inositol triphosphate receptors/calcium-operated channels induced by ma and la, we used pertussis toxin, u, and 2-apb, respectively. we demonstrated that preincubation with 100 ng/ml ptx for 16 h did not inhibit the calcium influx induced either by ma or la fatty acids in buvecs, which suggests that gαi/o does not participate in this type of response. the mobilization of calcium by la was reduced by 4 μm u but the effect of ma was not affected by treatment with the plc inhibitor. 2-apb reduced both the ma- and la-mediated increases in calcium by and 23 %, respectively. there is some evidence that fatty acids can directly and indirectly activate the l-type calcium channels in hek cos- and rat insulinoma ins-1e cells in vitro. however, the participation of ltccs in the calcium increase induced by ma or la in buvecs has not been evaluated. therefore, we investigated whether these channels participate in the calcium increase induced by ma or la stimulation. our results show that the ltcc blocker nifedipine inhibited the increases in calcium induced by ma and la by and 22 %, respectively. these results suggest that ltccs participate in the increases in calcium caused by saturated and unsaturated fatty acids in buvecs. ffar1/gpr expression and activity to examine ffar1/gpr protein expression in buvecs, we used western blotting and immunofluorescence assays directed against the conserved epitope of bovine gpr as described elsewhere. an immunoblotting analysis of the total protein extracts of buvecs showed a band with an apparent molecular size of 23-kda, lower than expected but similar to that observed in lovo cells, which were used as the positive control, as suggested by the manufacturer of the anti-gpr antibody. the same antibody was used for the immunofluorescence assays of buvecs, which detect the internal region of human gpr and therefore the intracellular and plasma membrane distribution. to confirm that the ffar1/gpr protein is in its active form in buvecs, we also measured the calcium signal separately with two known ffar1/gpr agonists, tak- and gw. tak- increased the intracellular calcium in a dose-dependent manner, whereas gw did not change the calcium signal at either low or high concentrations.fig. protein expression and functional detection of ffar1/gpr in buvec monolayers. a western blot of bovine ffar1/gpr total protein extract from lovo cells was used as the positive control for two buvec isolates. 50 μg of total protein was loaded onto a 6 % polyacrylamide gel. b ffar1/gpr immunofluorescence. left image, nuclear signals of cells incubated with propidium iodide; inset image: only the secondary antibody was used, as the negative control, bar = 20 μm. middle image, ffar1/gpr detected with rabbit anti-gpr antiserum and an anti-rabbit igg secondary antibody. right image, nuclear signals and gpr images are superimposed, bar = 20 nm. c representative time course of the fura-2/am ratio in buvecs exposed to 1 % dmso or 100 μm tak- at the time indicated by the arrow. d cells were exposed to dmso or tak- the area under the curve was calculated during the first 100 s after exposure to the stimulus. the data are expressed as the means ± sem of at least three independent experiments. **p < , ***p <  la increases nitric oxide production to assess whether nefas affect no production in the endothelium, we exposed buvecs to ma, sa, pa, oa, or la and evaluated the cells for no production using daf-fm signals. only la significantly increased the daf-fm fluorescence intensity in buvec monolayers. as illustrated in fig. 4a, the daf-fm signal increased approximately -fold in la-exposed buvecs compared with the signal in cells treated with vehicle. the increase in no induced by la was reduced by 25 % when the cells were treated with 100 μm l-name, a potent endothelial nitric oxide synthase inhibitor. this indicates that la activates enos in primary bovine endothelial cells. treatment with bapta, egta, or bapta plus egta reduced the increase in no induced by la by and 86 %, respectively. in contrast, neither 4 μm u 10 μm gw 100 μm 2-apb, 20 μm nifedipine, nor 100 ng/ml ptx reduced the la-mediated increase in no.fig. nefa effects on nitric oxide production in buvecs. a basal daf fluorescence, and after treatment with  % ethanol, 300 μm myristic acid, 300 μm stearic acid,  % dmso, 300 μm palmitic acid, 300 μm oleic acid, or 300 μm linoleic acid for 5 min at 37 °c. b daf fluorescent signal after incubation with 300 μm la for 5 min, normalized to the signal without fatty acids. the cells were incubated with bapta, egta, or bapta + egta before treatment with la. c effect of l-name and gw on the la-induced increase in the daf signal. basal fluorescence. daf fluorescence is expressed in arbitrary units. each bar represents the mean ± sem of at least three independent experiments. *p < , **p < , ***p <  la but not ma increases icam- and il- mrna expression because only la effectively increased enos activity in buvecs, we tested whether la also affects icam- or il- expression. only la induced the expression of icam- and il- mrnas in buvecs, and la-induced il- expression was significantly inhibited by gw however, the expression of icam- was not reduced by treatment with the ffar1/gpr antagonist.fig. nefa effects on icam- and il- expression in buvecs. cells were treated with  % ethanol, 300 μm myristic acid,  % dmso, 300 μm oleic acid, or 300 μm linoleic acid for 3 h and their rnas were then isolated. some cells were treated with 10 μm gw or 4 μm u for 30 min before the la treatment. the expression of icam- and il- was analyzed with real-time pcr using specific primers. relative abundance = 2–Δct, where Δct = cycle treshold icam- or ct il- minus cycle treshold sdha. the data are expressed as the means ± sem of three experiments. *p <  discussion the nefa levels in blood plasma are particularly elevated during the transition period in dairy cattle, when lipids are strongly mobilized from the adipose tissues to the bloodstream, with plasma concentrations increasing from the basal level of  mm to –2 mm. until now, the involvement of g-coupled fatty acid receptors, such as ffar1/gpr in the nefa-mediated calcium response in primary endothelial cells has been unclear. this study has demonstrated that single nefas alone, at low concentrations of  mm, increase the intracellular calcium response in primary bovine endothelial cells, inducing different molecular pathways involved in the endothelial cell activation process. in naturally occurring acute systemic coliform mastitis linoleic acid and oleic acid were the most abundant nefa, suggesting that specific fatty acids can be involved in inflammation and innate immunity, and to be relevant as risk factors during the transition period. different studies have reported the potency with which ma and la bind to human ffar1/gpr in transfected cho and hek cells by measuring the calcium signal. for ma, a ec value has been described in these cells, close to 7 μm, but for la, the ec values ranged from to  μm. in contrast, the ec values for ma and la were and  μm, respectively, in a reporter cell line based on hela cells transfected with hgpr. these results are quite similar to those observed in the present study, especially regarding the stimulatory potency of la. because many of the effects of nefas on primary bovine endothelial cells could be attributed to bgpr including increased intracellular calcium influx, the presence of this specific nefa receptor was assessed in buvecs in this study. we detected ffar1/gpr expression in buvecs, and also evaluated its effect on the calcium flux using the agonist tak- in our in vitro primary bovine endothelial cell system. there is some evidence that ffar1/gpr is expressed in bovine cells and bovine ffar1/gpr has been shown to share 84 % nucleotide sequence identity with the human isoform. previous studies have examined the expression of gpr in endothelial cells from monkey brain tissues, neuromicrovascular cells from rodent tissues, and human cardiac endothelial cells. to the best of our knowledge, this work is the first to report the expression of gpr in primary bovine endothelial cells. our data also suggest that the ma-mediated increase in calcium occurs when ma binds to bgpr because it was clearly inhibited by gw a potent ffar1/gpr antagonist. in contrast, the increase in calcium induced by la does not involve this receptor, according to our antagonist experiments. we also observed that ip receptors/calcium-operated channels and ltccs seem to participate in the cellular responses to ma and la, respectively. others have shown, that oa increased the calcium signal in hek cells transiently transfected with the α subunits of the ltccs. the pathway of calcium entry via the ltccs induced by nefas is evidently cell-type dependent. ltccs were also associated with the increase in calcium mediated by ffar1/gpr activation that was induced by the saturated fatty acid pa in rat β-cell line ins-1e, by the unsaturated fatty acid oa in islet β-cells from rats, and by la in primary cultured chicken hepatocytes. our examination of the cellular calcium flux suggests that ma, but not la, recognizes ffar1/gpr in buvecs. the cacna2d gene encodes the alpha 2/delta subunit which is an accessory part of the voltage-gated calcium channel family and considered to be an important candidate protein influencing bovine mastitis. although many publications have reported ltcc activity in bovine immune cells, there is not yet any evidence that these channels are expressed in bovine endothelial cells. in the present study, nifedipine affected buvecs, suggesting the participation of ltccs. it has also been recently demonstrated that the increase in calcium caused by endothelin- is ltcc-dependent in cultured bovine corneal endothelial cells. we also evaluated the no response in nefa-exposed bovine endothelium, because no is a well-known indicator of inflammation. all nefas were evaluated, but only la increased no in the endothelial cells. this response was dependent on both calcium and enos. there is evidence that enos activity is dependent on ca2+/calmodulin, and it is probable that the increase in no is a secondary effect after the interaction of enos with ca+2/calmodulin. both no and enos actively participate in the regulation of vascular tone and are therefore important intracellular enzymes, especially in microvascular hyperpermeability. there is some evidence that increased levels of no in endothelial cells produce proinflammatory profiles, which might lead to metabolic diseases. la induces reactive oxygen species and the overproduction of no after an increase in calcium, causing peroxynitration in endothelial cells. nf-κb, a crucial transcription factor, is then activated by peroxynitrite, a potent oxidative agent, inducing the expression of adhesion molecules and chemokines, such as il-. we also demonstrated that la, but not ma, induces icam-1- and il-8-expression, suggesting an active role for no in this response. in bovine endothelial cell cultures, high concentrations of total nefas might increase icam- mrna and protein expression. the la-induced adhesion molecules and chemokines may participate in leukocyte-endothelial cell interactions, facilitating transendothelial migration, and may play a role in the onset of a proinflammatory process during the transition period in dairy cattle. massive lipid mobilization leading to an increase in plasma nefas is observed in cows around parturition, causing an increased incidence of metabolic and infectious diseases, including laminitis, endometritis, and mastitis. the exposure of bovine endothelial cells to nefas increases the expression of the proinflammatory cytokines il- and il- and the adhesion molecules icam- and vcam-. it has also been postulated that the excessive accumulation of nefas during chronic inflammatory responses might increase tnfα, il- and il-1β expression by activating nf-κb in adipocytes and macrophages. these cytokines are thought to link the metabolic state and the inflammatory response, usually in the postpartum period within the uteri of cows. there is evidence that endothelial calcium participates in the transendothelial lymphocyte migration caused by icam-. moreover, an intracellular calcium increase has been related with icam- expression in huvec. however, in buvecs, intracellular calcium increase could be not directly associated with the expression of icam- or il- consistently with these assumption ma increased calcium flux was not sufficient to increase the expression of these molecules and the inhibition of plc did not reduce the expression of icam- and il- induced by la. our data also demonstrate that the individual nefas have different effects on endothelial cell activity and diverse mechanisms of action. la induces the expression of icam- independently of ffar1/gpr or plc activity, although the expression of il- is associated with the activation of ffar1/gpr in buvecs. in contrast, our results showed that no was induced by la, in a rather g-protein-coupled-receptor-independent process. there is some evidence that these receptors need not bind to a g-protein in the presence of high agonist concentrations, which thereby alternatively activate the erk/mapk pathway. it has been suggested that la activates the phosphoinositide 3-kinase, erk1/ and p- mapk pathways in vascular endothelial cells. in our laboratory, we have recently shown that the increase in no caused by la is p38-dependent. for this reason, it is possible that la activates ffar1/gpr or another receptor in a g-protein-independent manner. for instance, we cannot exclude the possibility that the gpr receptor recognizes la by another mechanism, or that la is recognized by gpr a medium-chain fatty acid receptor, or cd a fatty acid transporter protein. only cd expression has been demonstrated in endothelial cells. it has also been proposed that la mobilizes intracellular calcium via an la metabolite that induces intracellular mitochondrial calcium fluxes.  we have demonstrated for the first time in primary bovine endothelial cells that gpr is involved in the increase in calcium induced by ma, and conversely, that the la calcium flux is not associated with this receptor. we have also demonstrated the participation of ip receptors/calcium-operated channels and ltccs in the cellular responses to ma and la. we observed ffar1/gpr expression in the bovine endothelium and confirmed its activity using its agonist, tak- only la increased no levels, and this was independent of ffar1/gpr only la induced the expression of icam- and il- and this phenomenon is a ffar1/gpr40- dependent cellular process. these results indicate that the responses of endothelial cells to individual nefas are highly variable. recently, it has been demonstrated that there is an association of la enhancement in plasma and milk in naturally occurring acute systemic coliform mastitis. therefore, the increase in total nefas that is described as risk factor for the onset of proinflammatory process that occur during transition periods in cattle, should be revised carefully in the future. further studies are required to determine whether other individual nefas participate in the bovine proinflammatory responses in vivo during the transition period and in other nefa-dependent disorders in cattle. 
the aim of this study was to assess the effects of nefas on the intracellular calcium influx, nitric oxide production, and icam- and il- expression in primary bovine umbilical vein endothelial cells. myristic, palmitic, stearic, oleic and linoleic acid rapidly increased ca2+i. nonesterified fatty acids are involved in proinflammatory processes in cattle, including in the increased expression of adhesion molecules in endothelial cells. the calcium response to all tested nefas showed an extracellular calcium dependence and only the la response was significantly inhibited until the intracellular calcium was chelated. only the calcium response to ma was significantly reduced by gw a selective g-protein-coupled free fatty acid receptor antagonist. these findings strongly suggest that each individual nefa stimulates endothelial cells in a different way, with clearly different effects on intracellular calcium mobilization, no production, and il- and icam- expression in primary buvecs. the ec values for ma and la were 125 μm and 37 μm, respectively, and the ma and la effects were dependent on calcium release from the endoplasmic reticulum stores and on the l-type calcium channels.
 nonesterified fatty acids are involved in proinflammatory processes in cattle, including in the increased expression of adhesion molecules in endothelial cells. however, the mechanisms underlying these effects are still unknown. the aim of this study was to assess the effects of nefas on the intracellular calcium influx, nitric oxide production, and icam- and il- expression in primary bovine umbilical vein endothelial cells.  myristic, palmitic, stearic, oleic and linoleic acid rapidly increased ca2+i. the calcium response to all tested nefas showed an extracellular calcium dependence and only the la response was significantly inhibited until the intracellular calcium was chelated. the ec values for ma and la were 125 μm and 37 μm, respectively, and the ma and la effects were dependent on calcium release from the endoplasmic reticulum stores and on the l-type calcium channels. only the calcium response to ma was significantly reduced by gw a selective g-protein-coupled free fatty acid receptor antagonist. we also detected a functional ffar1/gpr protein in buvecs by using western blotting and the ffar1/gpr agonist tak- only la increased the cellular nitric oxide levels in a calcium-dependent manner. la stimulation but not ma stimulation increased icam- and il-8-expression in buvecs. this effect was inhibited by gw an antagonist of ffar1/gpr but not by u- a phospholipase c inhibitor.  these findings strongly suggest that each individual nefa stimulates endothelial cells in a different way, with clearly different effects on intracellular calcium mobilization, no production, and il- and icam- expression in primary buvecs. these findings not only extend our understanding of nefa-mediated diseases in ruminants, but also provide new insight into the different molecular mechanisms involved during endothelial cell activation by nefas. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords nonesterified fatty acidprimary bovine endothelial cellinterleukin 8intercellular adhesion molecule 1calcium fluxfondecyt312025511207181151047loaiza anitsi hidalgo maría a. burgos rafael a. fondef ideaid14i10050hidalgo maría a. issue-copyright-statement© the author 2016 