Regulation of Ca²⁺sensitization by PKC and rho proteins in ovine cerebral arteries: effects of artery size and age

Abstract

G protein-regulated Ca2+ sensitivity of vascular contractile proteins plays an important role in cerebrovascular reactivity. The present study examines the intracellular mechanisms that govern G protein-regulated Ca2+ sensitivity in cerebral arteries of different size and age. We studied β-escin-permeabilized segments of common carotid, basilar, and middle cerebral arteries from nonpregnant adult and near-term fetal sheep. Activation of protein kinase C (PKC) by (−)-indolactam V or a phorbol ester produced receptor-independent increases in Ca2+ sensitivity. Such increases were more marked in immature arteries and were inversely correlated with artery size in both mature and immature arteries. However, inhibitors of PKC did not significantly affect increases in Ca2+ sensitivity in responses to either serotonin (5-hydroxytryptamine, 5-HT) or guanosine 5′- O-(3-thiotriphosphate) (GTPγS). Alternatively, deactivation of rho p21, a small G protein associated with Rho kinase, by exotoxin C3 fully prevented increases in Ca2+ sensitivity in responses to 5-HT or GTPγS in both adult and fetal arteries of all types. Neither inhibitors of PKC nor exotoxin C3 altered baseline Ca2+ sensitivity. We conclude that patterns of receptor- and/or G protein-mediated modulation of Ca2+ sensitivity are dependent on an intracellular pathway that involves activation of small G proteins and Rho kinase. In contrast, PKC has little, if any, role in agonist-induced Ca2+ sensitization under the present experimental conditions.

Evidence That Rho-Kinase Activity Contributes to Cerebral Vascular Tone In Vivo and Is Enhanced During Chronic Hypertension

“…12,13 Pharmacological activation of PKC, eg, by using phorbol esters, is known to be a powerful cerebral vasoconstrictor stimulus in vitro 4,[23][24][25] and in vivo 26 -28 because of increased Ca 2ϩ sensitivity of vascular contractile elements. We confirmed that activation of PKC is a strong constrictor stimulus in the basilar artery, in that phorbol 12,13-dibutyrate produced marked vasoconstriction that could be blocked by pretreating the artery with either of two relatively selective PKC inhibitors, calphostin C or Ro 31-8220.…”

“…Such a conclusion is conceptually consistent with recent findings in isolated and permeabilized ovine cerebral arteries, in which agonist-induced Ca 2ϩ sensitization could be prevented by inactivation of Rho (using exotoxin C3) but was not affected by inhibition of PKC. 4 Thus, although activation of PKC and Rho-kinase may both lead to increased vascular tone through Ca 2ϩ sensitization, it appears that only the latter mechanism is normally active in the basilar artery under resting conditions. Moreover, the finding that calphostin C had virtually no effect on baseline diameter of the basilar artery in SHR or WKY rats suggests that the influence of PKC activity on cerebral artery tone under basal conditions in vivo remains insignificant during chronic hypertension.…”

“…2 Rho is known to be activated by numerous trimeric G proteins, and Rho/Rho-kinase is therefore expected to play a fundamental role in vascular signal transduction during responses to vasoconstrictor agonists 2 and also stretch. 3 The use of bacterial exotoxin C3, which deactivates Rho, provided recent indirect evidence that activation of Rho (and thus potentially Rho-kinase) modulates Ca 2ϩ sensitivity in permeabilized cerebral arteries in vitro, 4 but the role of Rho-kinase in regulating intact cerebral vessel tone in vivo is not known, nor are effects of cerebrovascular disease states on Rhokinase function.…”

Mechanisms involved in carbachol‐induced Ca2+ sensitization of contractile elements in rat proximal and distal colon

“…However, the extent of the contribution of the two pathways in Ca 2 þ sensitization differs with the type of smooth muscle and the species of animals. Utilizing C3 exoenzyme, a specific inhibitor of rho protein, and Y-27632 and HA 1077, rho kinase inhibitors, the involvement of a rho-rho kinase pathway in agonist-induced Ca 2 þ sensitization was suggested in the rabbit aorta (Kokubu et al, 1995;Uehara et al, 1997), pulmonary artery (Fu et al, 1998), portal vein (Fu et al, 1998) and trachea (Iizuka et al, 1999), guinea pig vas deferens and ovine cerebral artery (Akopov et al, 1998). In contrast, a protein kinase C pathway was proposed to mediate agonistinduced Ca 2 þ sensitization in the rabbit femoral (Gailly et al, 1997), rabbit mesenteric artery (Nishimura et al, 1992), rabbit portal vein (Brozovich, 1995), ferret portal vein (Lee et al, 1999) and rat tail artery (Weber et al, 2000), as based on the inhibitory effects of protein kinase C inhibitors, GF109203X and chelerythrine, and an inhibitory peptide of protein kinase C. However, only one report, in canine tracheal smooth muscle, indicates the involvement of both pathways in agonist-induced Ca 2 þ sensitization in the same tissue (Iizuka et al, 1997).…”

Role of Protein Kinase Network in Excitation-Contraction Coupling in Smooth Muscle Cell

“…PKC has been shown to be involved in force maintenance in human airways (Rossetti et al, 1995). However, other studies do not implicate PKC in agonistinduced Ca 2+ sensitization and point to other effectors such as the small GTP-binding protein p21rho (Akopov et al, 1998;Itoh et al, 1994;Otto et al, 1996;Yoshii et al, 1999). Our recent studies have shown that PKC contributes to the sustained phase, but not to the initial phase, of cholinergic-induced contraction in rat airways (Mbikou et al, 2006).…”