Prolonged bed rest impairs rapid CPI-17 phosphorylation and contraction in rat mesenteric resistance arteries to cause orthostatic hypotension.

Prolonged bed rest (PBR) causes orthostatic hypotension (OH). Rapid constriction of splanchnic resistance arteries in response to a sudden increase in sympathetic tone contributes to the recovery of orthostatic arterial pressure upon standing. However, the molecular mechanism of PBR-induced dysfunction in arterial constriction is not fully understood. Previously, we showed that CPI-17, a regulatory protein for myosin phosphatase, mediates α1A-adrenergic receptor-induced rapid contraction of small mesenteric arteries. Here, we tested whether PBR associated with OH affects the α1-adrenergic receptor-induced CPI-17 signaling pathway in mesenteric arteries using rats treated by head-down tail-suspension hindlimb unloading (HDU), an experimental OH model. In normal anesthetized rats, mean arterial pressure (MAP) rapidly reduced upon 90° head-up tilt from supine position and then immediately recovered without change in heart rate, suggesting a rapid arterial constriction. On the other hand, after a 4-week HDU treatment, the fast orthostatic MAP recovery failed for 1 min. Alpha1A subtype-specific antagonist suppressed the orthostatic MAP recovery with a small decrease in basal blood pressure, whereas non-specific α1-antagonist prazosin strongly reduced both basal MAP and orthostatic recovery. The HDU treatment resulted in 68% reduction in contraction in parallel with 83% reduction in CPI-17 phosphorylation in denuded mesenteric arteries 10 s after α1-agonist stimulation. The treatment with either Ca2+-release channel opener or PKC inhibitor mimicked the deficiency in HDU arteries. These results suggest that an impairment of the rapid PKC/CPI-17 signaling pathway downstream of α1A-adrenoceptors in peripheral arterial constriction, as an end organ of orthostatic blood pressure reflex, is associated with OH in prolonged bed rest patients.

Size-dependent heterogeneity of contractile Ca2+ sensitization in rat arterial smooth muscle.

Each segment along arterial vessels adapts to different circumstances, including blood pressure and sympathetic innervation. PKC and Rho-associated kinase (ROCK) Ca(2+)-sensitizing pathways leading to myosin phosphatase inhibition are critically involved in α(1)-adrenoceptor-mediated vascular smooth muscle contraction in distinctive time-dependent manners. We tested whether the amplitude and time course of each pathway varies dynamically between arterial segments. Using pharmacological approaches, we determined the time-dependent roles of Ca(2+) release, Ca(2+) influx, PKC and ROCK in α(1)-agonist-induced contraction and phosphorylation of key proteins in denuded rat small mesenteric artery, midsized caudal artery and thoracic aorta. SR Ca(2+) release and voltage-dependent Ca(2+) influx were essential for the initial rising and late sustained phases, respectively, of phenylephrine-induced contraction, regardless of arterial size. In small mesenteric arteries, α(1A)-subtype-specific antagonists and inhibitors of PKC, but not ROCK, markedly reduced the initial and late phases of contraction in a non-additive manner and suppressed phosphorylation of myosin light chain (MLC) and CPI-17, but not myosin targeting subunit of myosin light chain phosphatase (MYPT1). In aorta, an α(1D)-specific antagonist reduced both the initial and late phases of contraction with a significant decrease in MLC but not CPI-17 or MYPT1 phosphorylation. ROCK inhibitors, but not PKC inhibitors, suppressed the sustained phase of contraction with a decrease in MLC and MYPT1 phosphorylation in the aorta. The effect of ROCK inhibitors was additive with the α(1D)-antagonist. The results for midsized arteries were intermediate. Thus, the PKC-CPI-17 Ca(2+)-sensitizing pathway, which is dependent on PKC subtype and a Ca(2+)-handling mechanism, and is downstream of α(1A) receptors, plays a major role in α(1)-agonist-induced contraction of small resistance arteries in the splanchnic vascular beds. The effect of PKC and ROCK increases and decreases, respectively, with decreasing arterial size.

Nitric oxide-induced biphasic mechanism of vascular relaxation via dephosphorylation of CPI-17 and MYPT1.

Nitric oxide (NO) from endothelium is a major mediator of vasodilatation through cGMP/PKG signals that lead to a decrease in Ca(2+) concentration. In addition, NO-mediated signals trigger an increase in myosin light chain phosphatase (MLCP) activity. To evaluate the mechanism of NO-induced relaxation through MLCP deinhibition, we compared time-dependent changes in Ca(2+), myosin light chain (MLC) phosphorylation and contraction to changes in phosphorylation levels of CPI-17 at Thr38, RhoA at Ser188, and MYPT1 at Ser695, Thr696 and Thr853 in response to sodium nitroprusside (SNP)-induced relaxation in denuded rabbit femoral artery. During phenylephrine (PE)-induced contraction, SNP reduced CPI-17 phosphorylation to a minimal value within 15 s, in parallel with decreases in Ca(2+) and MLC phosphorylation, followed by a reduction of contractile force having a latency period of about 15 s. MYPT1 phosphorylation at Ser695, the PKG-target site, increased concurrently with relaxation. Phosphorylation of RhoA, MYPT1 Thr696 and Thr853 differed significantly at 5 min but not within 1 min of SNP exposure. Inhibition of Ca(2+) release delayed SNP-induced relaxation while inhibition of Ca(2+) channel, BK(Ca) channel or phosphodiesterase-5 did not. Pretreatment of resting artery with SNP suppressed an increase in Ca(2+), contractile force and phosphorylation of MLC, CPI-17, MYPT1 Thr696 and Thr853 at 10 s after PE stimulation, but had no effect on phorbol ester-induced CPI-17 phosphorylation. Together, these results suggest that NO production suppresses Ca(2+) release, which causes an inactivation of PKC and rapid CPI-17 dephosphorylation as well as MLCK inactivation, resulting in rapid MLC dephosphorylation and relaxation.

Ca2+-dependent rapid Ca2+ sensitization of contraction in arterial smooth muscle.

Ca(2+) ion is a universal intracellular messenger that regulates numerous biological functions. In smooth muscle, Ca(2+) with calmodulin activates myosin light chain (MLC) kinase to initiate a rapid MLC phosphorylation and contraction. To test the hypothesis that regulation of MLC phosphatase is involved in the rapid development of MLC phosphorylation and contraction during Ca(2+) transient, we compared Ca(2+) signal, MLC phosphorylation, and 2 modes of inhibition of MLC phosphatase, phosphorylation of CPI-17 Thr38 and MYPT1 Thr853, during alpha(1) agonist-induced contraction with/without various inhibitors in intact rabbit femoral artery. Phenylephrine rapidly induced CPI-17 phosphorylation from a negligible amount to a peak value of 0.38+/-0.04 mol of Pi/mol within 7 seconds following stimulation, similar to the rapid time course of Ca(2+) rise and MLC phosphorylation. This rapid CPI-17 phosphorylation was dramatically inhibited by either blocking Ca(2+) release from the sarcoplasmic reticulum or by pretreatment with protein kinase C inhibitors, suggesting an involvement of Ca(2+)-dependent protein kinase C. This was followed by a slow Ca(2+)-independent and Rho-kinase/protein kinase C-dependent phosphorylation of CPI-17. In contrast, MYPT1 phosphorylation had only a slow component that increased from 0.29+/-0.09 at rest to the peak of 0.68+/-0.14 mol of Pi/mol at 1 minute, similar to the time course of contraction. Thus, there are 2 components of the Ca(2+) sensitization through inhibition of MLC phosphatase. Our results support the hypothesis that the initial rapid Ca(2+) rise induces a rapid inhibition of MLC phosphatase coincident with the Ca(2+)-induced MLC kinase activation to synergistically initiate a rapid MLC phosphorylation and contraction in arteries with abundant CPI-17 content.

Agonist- and depolarization-induced signals for myosin light chain phosphorylation and force generation of cultured vascular smooth muscle cells.

Phosphorylation of myosin light chain (MLC) and contraction of differentiated smooth muscle cells in vascular walls are regulated by Ca2+ -dependent activation of MLC kinase, and by Rho-kinase- or protein-kinases-C-dependent inhibition of MLC phosphatase (MLCP). We examined regulatory pathways for MLC kinase and MLCP in cultured vascular smooth muscle cells (VSMCs), and for isometric force generation of VSMCs reconstituted in collagen fibers. Protein levels of RhoA, Rho-kinase and MYPT1 (a regulatory subunit of MLCP) were upregulated in cultured VSMCs, whereas a MLCP inhibitor protein, CPI-17, was downregulated. Endothelin-1 evoked a steady rise in levels of Ca2+, MLC phosphorylation and the contractile force of VSMCs, whereas angiotensin-II induced transient signals. Also, Thr853 phosphorylation of MYPT1 occurred in response to stimuli, but neither agonist induced phosphorylation of MYPT1 at Thr696. Unlike fresh aortic tissues, removal of Ca2+ or addition of voltage-dependent Ca2+ -channel blocker did not inhibit contractions of reconstituted VSMC fibers induced by agonists or even high concentrations of extracellular K+ ions. Inhibitors of Ins(1,4,5)P3-receptor and Rho-kinase antagonized agonist-induced or high-K+ -induced contraction in both reconstituted fibers and fresh tissues. These results indicate that both Ins(1,4,5)P3-induced Ca2+ release and Rho-kinase-induced MYPT1 phosphorylation at Thr853 play pivotal roles in MLC phosphorylation of cultured VSMCs where either Ca2+ -influx or CPI-17-MLCP signaling is downregulated.