Ca2+ signalling in rat vascular smooth muscle cells: a role for protein kinase C at physiological vasoconstrictor concentrations of vasopressin

Arg8-vasopressin (AVP) is a peptide hormone that is released from the posterior pituitary gland into the systemic circulation in response to a decrease in blood pressure or an increase in plasma osmolality. A baroreceptor response to a large decrease in blood volume may cause plasma concentrations of AVP to increase from a few picomolar to a few hundred picomolar, resulting in arterial constriction and increased peripheral vascular resistance. The potent vasoconstrictor actions of AVP have been attributed to its activation of phospholipase C and the resulting release of Ca2+ from intracellular stores within vascular smooth muscle cells. A7r5 cells are a smooth muscle cell line derived from embryonic rat aorta (Kimes & Brandt, 1976). As in other vascular smooth muscle preparations, stimulation of A7r5 cells with AVP results in an increase in [Ca2+]i by release of Ca2+ from intracellular stores and increased Ca2+ influx (Byron & Taylor, 1995). However, the concentration of AVP required for half-maximal release from intracellular Ca2+ stores is approximately 5 nM, much too high to account for the vasoconstrictor effects of the picomolar concentrations of AVP found in the systemic circulation. A7r5 cells exhibit spontaneous Ca2+ spikes in the absence of AVP. The mechanism by which this Ca2+-spiking activity is generated involves activation of L-type voltage-sensitive Ca2+ channels and is independent of the release of Ca2+ from intracellular stores (Byron & Taylor, 1993). Ca2+ spiking in vascular smooth muscle may lead to rhythmic vasomotion of small resistance arteries and arterioles. Vasomotion has been observed in vivo as well as in a number of arterial preparations in vitro and is believed to be important in the regulation of regional tissue blood supply (Nicoll & Webb, 1955) as well as contributing to peripheral vascular resistance (Gratton et al. 1998). Most of our knowledge of spontaneous vasomotor activity is derived from studies of the contraction of vessel segments and strips of vascular tissue. Information about the mechanisms involved in generating and regulating this activity is limited because of the presence of multiple cell types in tissue preparations and an inability to dissociate changes in [Ca2+]i from changes in sensitivity of the contractile mechanisms. However, in recent years, measurements of [Ca2+]i in populations of cultured vascular smooth muscle have revealed that these cells, in isolation from other cell types, retain the ability to spontaneously generate transient increases in [Ca2+]i (Ca2+ spikes) that correspond to the spontaneous electrical activity of the plasma membrane (Weissberg et al. 1989; Byron & Taylor, 1993). We have previously shown that the frequency of Ca2+ spiking in A7r5 cells is exquisitely sensitive to concentrations of AVP found in the systemic circulation (Byron, 1996). The present study examines in more detail the signal transduction pathways involved in the stimulation of Ca2+ spiking by AVP, focusing specifically on the role of PKC. The PKC family of lipid-regulated serine/threonine kinases comprises at least eleven identified isoforms of PKC, which have been grouped into three classes. Conventional PKCs (α, βI, βII and γ) are activated by Ca2+ and/or by diacylglycerol (DAG) and phorbol esters. Novel PKCs (δ, e, θ and η) are Ca2+-independent isoforms that are also activated by DAG and phorbol esters, whereas atypical PKCs (l/λ, ζ) are independent of both Ca2+ and DAG/phorbol esters (Ron & Kazanietz, 1999). Activation of conventional and novel PKCs by DAG or phorbol esters is thought to involve a conformational change that leads to a subcellular redistribution or translocation of the PKC from cytosolic to membrane compartments. It is well known that activation of G-protein-coupled receptors by vasoconstrictor hormones, such as AVP, angiotensin II, endothelin and noradrenaline, leads to activation of PKC. Early studies implicated PKC in mitogenic responses (Rozengurt, 1986) as well as in the regulation of vascular smooth muscle contractility (Rasmussen et al. 1984). With the more recent identification of multiple PKC isoforms with distinct tissue distributions and regulatory properties (Hofmann, 1997; Ron & Kazanietz, 1999), the specific PKC isoforms that are activated by vasoconstrictors and their roles in specific signalling pathways are just beginning to be characterized. Our findings suggest a central role for PKC in the novel signalling pathway linking physiological concentrations of AVP with an increase in the frequency of Ca2+ spiking.