BACKGROUND
ca2+-dependent phosphorylation of the myosin regulatory light chain  is accepted as the primary mechanism regulating contraction of smooth muscle in response to excitatory stimuli  <cit> .
however, this myosin-linked phosphorylation mechanism does not fully explain all observations.
of particular physiological interest is relaxation induced in activated tissues by no or no-donors that increase .
in both vascular smooth muscle  <cit>  and corpus cavernosum  <cit> , there are two mechanism for no donors to reduce tone.
1) no donors can reduce in myoplasmic , which decreases mrlc phosphorylation, a process termed "deactivation."
deactivation is demonstrated when a decline in force is associated with a dependence of force on mrlc phosphorylation similar to that observed with contractile agonists alone.
2) no donors can also reduce force without reductions in myoplasmic  or mrlc phosphorylation, a mechanism termed "force suppression"  <cit> .
force suppression is demonstrated when a decline in force is associated with a rightward shift in the dependence of force on mrlc phosphorylation similar to that observed with contractile agonists alone  <cit> .
we hypothesized that a thin filament mechanism, specifically ser <dig> phosphorylation of heat shock protein was the mediator of force suppression  <cit> .
the human umbilical artery was found to not express hsp <dig> and not exhibit no dependent relaxation  <cit> , suggesting a possible linkage between hsp <dig> and no dependent relaxation.
the rabbit bladder is a phasic urogenital smooth muscle that does not relax in response to no.
we therefore hypothesized that the rabbit bladder could be another test of the role of hsp <dig> in force suppression.
we therefore tested 1) whether hsp <dig> is expressed in rabbit bladder and 2) whether force suppression occurs with relaxing agents in the rabbit bladder.
methods
tissues
male new zealand white rabbits  were euthanized by halothane inhalation according to an iacuc approved protocol.
the urinary bladder was isolated at 4°c in a bicarbonate-buffered krebs solution containing   <dig>  nacl,  <dig>  kcl,  <dig>  nahco <dig>   <dig>  kh2po <dig>   <dig>  cacl <dig>   <dig>  mgso4·7h2o, and  <dig>  d-glucose saturated with 95% o <dig> and 5% co <dig>  an incision was made from the bladder neck up to the dome following either the dorsal or ventral vasculature.
the bladder was pinned out with the mucosa facing down.
this protocol caused ridges to form from which strips were dissected from the abluminal surface.
histological examination showed that the cells were aligned in the longitudinal axis of the preparation in which length and force were measured .
the bladder strips were tied to the two posts on the apparatus using silk sutures; one post to a micrometer to change length, and the other to a ft <dig>  grass force transducer.
the length was incrementally increased until a constant force of  <dig> g was maintained, approximating lo.
the preparations responded with sustained contractions when exposed to  <dig> μm carbachol.
k+-depolarization elicited transient contractions diagnostic of a phasic smooth muscle  <cit> .
tissues exhibiting spontaneous oscillatory activity were excluded from the analysis.
a total of  <dig> tissues were included in the analysis.
mrlc and hsp <dig> phosphorylation
bladder strips treated pharmacologically and then frozen in  <dig> ml of acetone cooled with  <dig> ml crushed dry ice.
they were then slowly  thawed to room temperature to dehydrate the tissues, air dried and weighed.
the dry samples were homogenized in ground glass tissue homogenizers on ice in 1%  sodium dodecyl sulfate , 10%  glycerol,  <dig> % pefabloc ,  <dig>  % microcystin, and  <dig> mm dithiothreitol , and then centrifuged at  <dig>  × g for  <dig> min.
trichorloacetic acid was not included since it did not alter mrlc phosphorylation estimates.
serial dilutions  of homogenates in homogenization buffer were loaded onto 12% acrylamide/glycerol-urea slab gels for isoelectric focusing at  <dig> volts overnight on a ph  <dig> – <dig>  gradient for mrlc  <cit>  and a ph  <dig> – <dig>  gradient for hsp <dig>  <cit> .
gels were focused at  <dig> v constant voltage for  <dig> h at 8°c.
separated proteins were transferred to a nitrocellulose membrane by electroblotting in towbin's transfer buffer  at  <dig> ma constant current for  <dig> h at 8°c.
the membranes were first washed in a  <dig> % tris-buffered saline-tween solution .
the membranes were then blocked overnight in tbst containing 1% bovine serum albumin and  <dig> % sodium azide.
after rinsing in tbst, the membranes were incubated in either 1: <dig> anti-mrlc antibody  or 1: <dig> rabbit anti-hsp <dig>  antibody for  <dig> h. after rinsing in tbst, the membranes were incubated with a horseradish peroxidase conjugate secondary  for  <dig> h. after rinsing twice with tbst and once with tbs , antibodies were detected with enhanced chemiluminescence was scanned using a molecular dynamics laser densitometer and analyzed by nih image software.
the relative protein content was estimated assuming that antibody binding was the same for phospho- and dephospho-mrlc and corrections made for offset and saturation errors as described  <cit> .
RESULTS
carbachol  alone induced a sustained contraction that measured  <dig> ±  <dig> % of a maximal  carbachol contraction.
therefore,  <dig>  μm carbachol was used to test relaxing agents.
preliminary experiments showed that  <dig>  μm carbachol stimulated rabbit bladder did not relax with  <dig> μm nitroprusside .
carbachol  stimulated rabbit bladder relaxed when treated with 8-bromo-cgmp , forskolin , or isoprenaline .
the open circles in fig.
<dig> shows the steep dependence of force on mrlc phosphorylation when rabbit urinary bladder was activated with  <dig> – <dig> μm carbachol .
the filled symbols in fig.
<dig> show the dependence of force on mrlc phosphorylation when  <dig>  μm carbachol stimulated rabbit bladder were relaxed with 8-bromo-cgmp , forskolin , or isoprenaline .
these three treatments induced a dependence of force on mrlc phosphorylation that did not differ from that observed with carbachol alone , suggesting the relaxation occurred by deactivation.
if there had been force suppression, there would have been a rightward shift in the dependence of force on mrlc phosphorylation.
hsp <dig> immunostaining was low in rabbit bladder  compared to swine carotid .
overall, there was  <dig>  fold less hsp <dig> immunostaining in the bladder homogenates compared to swine carotid homogenates.
since this comparison was normalized on tissue dry weight, we also compared mrlc immunostaining from the same samples.
there was  <dig>  fold more mrlc immunostaining in the bladder homogenates compared to swine carotid homogenates .
when hsp <dig> immunostaining was normalized by mrlc immunostaining, there was  <dig> fold less hsp <dig> immunostaining in the bladder compared to the swine carotid immunostaining.
discussion
these data suggest that rabbit bladder does not express significant levels of hsp <dig>  and does not exhibit force suppression .
this result is consistent with the hypothesis that hsp <dig> is the mediator of force suppression  <cit> .
our results are reminiscent of data showing that umbilical vein does not express significant levels of hsp <dig> and does not relax to no donors  <cit> .
we think it most likely that the low level of hsp <dig> immunostaining is caused by hsp <dig> present in the vascular smooth muscle of the bladder vasculature.
however, we cannot rule out a low level of hsp <dig> expression in bladder smooth muscle.
we assumed that carotid and bladder have similar cellular mrlc concentration  so that mrlc immunostaining could be used to normalize hsp <dig> immunostaining.
the accuracy of this assumption is not that crucial given the relatively small amount of hsp <dig> immunostaining in the bladder .
we confirmed that the rabbit bladder does not relax to no donors.
however, there was a relaxation to 8-bromo-cgmp, a cell permeant cgmp analog, suggesting that the lack of response to no donors resides in the generation of cgmp, rather than a defect in the response to cgmp.
non-receptor activators of adenyl cyclase  also induced relaxation, suggesting that rabbit bladder can relax to increases in camp.
importantly, these relaxations cgmp and camp mediated relaxations were not associated with force suppression .
this is consistent with the hypothesis that hsp <dig> is involved in force suppression.
the relaxations in response to cgmp analogs and agents that increase  suggest that the relaxation is caused by deactivation mechanisms such as mechanisms that reduce i or possibly increase myosin phosphatase activity  <cit> .
CONCLUSIONS
these results suggest that some phasic smooth muscles, such as rabbit bladder, do not exhibit force suppression.
force suppression was found in corpus cavernosum  <cit> , a no responsive phasic smooth muscle.
unlike the human umbilical vein study  <cit> , we report that the lack of hsp <dig> is not only associated with a lack in relaxation, but also a lack of force suppression.
competing interests
the author declare that they have no competing interests.
authors' contributions
tb and jw performed the studies.
rm and cr conceived the study.
tb and cr drafted the study.

 ca2+-dependent phosphorylation of the myosin regulatory light chain is accepted as the primary mechanism regulating contraction of smooth muscle in response to excitatory stimuli. however, this myosin-linked phosphorylation mechanism does not fully explain all observations. of particular physiological interest is relaxation induced in activated tissues by no or no-donors that increase. in both vascular smooth muscle and corpus cavernosum, there are two mechanism for no donors to reduce tone. 1) no donors can reduce in myoplasmic, which decreases mrlc phosphorylation, a process termed "deactivation." deactivation is demonstrated when a decline in force is associated with a dependence of force on mrlc phosphorylation similar to that observed with contractile agonists alone. 2) no donors can also reduce force without reductions in myoplasmic or mrlc phosphorylation, a mechanism termed "force suppression". force suppression is demonstrated when a decline in force is associated with a rightward shift in the dependence of force on mrlc phosphorylation similar to that observed with contractile agonists alone. we hypothesized that a thin filament mechanism, specifically ser phosphorylation of heat shock protein was the mediator of force suppression. the human umbilical artery was found to not express hsp and not exhibit no dependent relaxation, suggesting a possible linkage between hsp and no dependent relaxation. the rabbit bladder is a phasic urogenital smooth muscle that does not relax in response to no. we therefore hypothesized that the rabbit bladder could be another test of the role of hsp in force suppression. we therefore tested 1) whether hsp is expressed in rabbit bladder and 2) whether force suppression occurs with relaxing agents in the rabbit bladder. methods tissues male new zealand white rabbits were euthanized by halothane inhalation according to an iacuc approved protocol. the urinary bladder was isolated at 4°c in a bicarbonate-buffered krebs solution containing nacl, kcl, nahco kh2po cacl mgso4·7h2o, and d-glucose saturated with 95% o and 5% co an incision was made from the bladder neck up to the dome following either the dorsal or ventral vasculature. the bladder was pinned out with the mucosa facing down. this protocol caused ridges to form from which strips were dissected from the abluminal surface. histological examination showed that the cells were aligned in the longitudinal axis of the preparation in which length and force were measured. the bladder strips were tied to the two posts on the apparatus using silk sutures; one post to a micrometer to change length, and the other to a ft grass force transducer. the length was incrementally increased until a constant force of g was maintained, approximating lo. the preparations responded with sustained contractions when exposed to μm carbachol. k+-depolarization elicited transient contractions diagnostic of a phasic smooth muscle. tissues exhibiting spontaneous oscillatory activity were excluded from the analysis. a total of tissues were included in the analysis. mrlc and hsp phosphorylation bladder strips treated pharmacologically and then frozen in ml of acetone cooled with ml crushed dry ice. they were then slowly thawed to room temperature to dehydrate the tissues, air dried and weighed. the dry samples were homogenized in ground glass tissue homogenizers on ice in 1% sodium dodecyl sulfate, 10% glycerol, % pefabloc, % microcystin, and mm dithiothreitol, and then centrifuged at × g for min. trichorloacetic acid was not included since it did not alter mrlc phosphorylation estimates. serial dilutions of homogenates in homogenization buffer were loaded onto 12% acrylamide/glycerol-urea slab gels for isoelectric focusing at volts overnight on a ph – gradient for mrlc and a ph – gradient for hsp. gels were focused at v constant voltage for h at 8°c. separated proteins were transferred to a nitrocellulose membrane by electroblotting in towbin's transfer buffer at ma constant current for h at 8°c. the membranes were first washed in a % tris-buffered saline-tween solution. the membranes were then blocked overnight in tbst containing 1% bovine serum albumin and % sodium azide. after rinsing in tbst, the membranes were incubated in either 1: anti-mrlc antibody or 1: rabbit anti-hsp antibody for h. after rinsing in tbst, the membranes were incubated with a horseradish peroxidase conjugate secondary for h. after rinsing twice with tbst and once with tbs, antibodies were detected with enhanced chemiluminescence was scanned using a molecular dynamics laser densitometer and analyzed by nih image software. the relative protein content was estimated assuming that antibody binding was the same for phospho- and dephospho-mrlc and corrections made for offset and saturation errors as described.  carbachol alone induced a sustained contraction that measured ± % of a maximal carbachol contraction. therefore, μm carbachol was used to test relaxing agents. preliminary experiments showed that μm carbachol stimulated rabbit bladder did not relax with μm nitroprusside. carbachol stimulated rabbit bladder relaxed when treated with 8-bromo-cgmp, forskolin, or isoprenaline. the open circles in fig. shows the steep dependence of force on mrlc phosphorylation when rabbit urinary bladder was activated with – μm carbachol. the filled symbols in fig. show the dependence of force on mrlc phosphorylation when μm carbachol stimulated rabbit bladder were relaxed with 8-bromo-cgmp, forskolin, or isoprenaline. these three treatments induced a dependence of force on mrlc phosphorylation that did not differ from that observed with carbachol alone, suggesting the relaxation occurred by deactivation. if there had been force suppression, there would have been a rightward shift in the dependence of force on mrlc phosphorylation. hsp immunostaining was low in rabbit bladder compared to swine carotid. overall, there was fold less hsp immunostaining in the bladder homogenates compared to swine carotid homogenates. since this comparison was normalized on tissue dry weight, we also compared mrlc immunostaining from the same samples. there was fold more mrlc immunostaining in the bladder homogenates compared to swine carotid homogenates. when hsp immunostaining was normalized by mrlc immunostaining, there was fold less hsp immunostaining in the bladder compared to the swine carotid immunostaining. discussion these data suggest that rabbit bladder does not express significant levels of hsp and does not exhibit force suppression. this result is consistent with the hypothesis that hsp is the mediator of force suppression. our results are reminiscent of data showing that umbilical vein does not express significant levels of hsp and does not relax to no donors. we think it most likely that the low level of hsp immunostaining is caused by hsp present in the vascular smooth muscle of the bladder vasculature. however, we cannot rule out a low level of hsp expression in bladder smooth muscle. we assumed that carotid and bladder have similar cellular mrlc concentration so that mrlc immunostaining could be used to normalize hsp immunostaining. the accuracy of this assumption is not that crucial given the relatively small amount of hsp immunostaining in the bladder. we confirmed that the rabbit bladder does not relax to no donors. however, there was a relaxation to 8-bromo-cgmp, a cell permeant cgmp analog, suggesting that the lack of response to no donors resides in the generation of cgmp, rather than a defect in the response to cgmp. non-receptor activators of adenyl cyclase also induced relaxation, suggesting that rabbit bladder can relax to increases in camp. importantly, these relaxations cgmp and camp mediated relaxations were not associated with force suppression. this is consistent with the hypothesis that hsp is involved in force suppression. the relaxations in response to cgmp analogs and agents that increase suggest that the relaxation is caused by deactivation mechanisms such as mechanisms that reduce i or possibly increase myosin phosphatase activity.  these results suggest that some phasic smooth muscles, such as rabbit bladder, do not exhibit force suppression. force suppression was found in corpus cavernosum, a no responsive phasic smooth muscle. unlike the human umbilical vein study, we report that the lack of hsp is not only associated with a lack in relaxation, but also a lack of force suppression. competing interests the author declare that they have no competing interests. authors' contributions tb and jw performed the studies. rm and cr conceived the study. tb and cr drafted the study. 
the lack of force suppression in the absence of hsp is consistent with the hypothesized role for hsp in the force suppression observed in tonic smooth muscles. this has been termed force suppression and been found to correlate with ser16-phosphorylation of heat shock protein. nitroglycerin can induce relaxation of swine carotid artery without sustained reductions in i or myosin regulatory light chain phosphorylation. we tested for the existence of this mechanism in a smooth muscle that is not responsive to nitric oxide. contraction was associated with a highly cooperative relation between mrlc phosphorylation and force such that very small increases in mrlc phosphorylation induced large increases in force. relaxation induced by 8-br-cgmp, forskolin, or isoprenaline did not shift the mrlc phosphorylation-force relation from that observed with carbachol alone, i.e.
 nitroglycerin can induce relaxation of swine carotid artery without sustained reductions in i or myosin regulatory light chain phosphorylation. this has been termed force suppression and been found to correlate with ser16-phosphorylation of heat shock protein. we tested for the existence of this mechanism in a smooth muscle that is not responsive to nitric oxide. methods isometrically mounted mucosa free rabbit bladder strips were contracted with carbachol and relaxed with 8-br-cgmp, forskolin, or isoprenaline.  contraction was associated with a highly cooperative relation between mrlc phosphorylation and force such that very small increases in mrlc phosphorylation induced large increases in force. relaxation induced by 8-br-cgmp, forskolin, or isoprenaline did not shift the mrlc phosphorylation-force relation from that observed with carbachol alone, i.e. there was no force suppression. hsp content was negligible.  the lack of force suppression in the absence of hsp is consistent with the hypothesized role for hsp in the force suppression observed in tonic smooth muscles. 