Vascular smooth muscle desensitization in rabbit epigastric and mesenteric arteries during hemorrhagic shock.

The decompensatory phase of hemorrhage (shock) is caused by a poorly defined phenomenon termed vascular hyporeactivity (VHR). VHR may reflect an acute in vivo imbalance in levels of contractile and relaxant stimuli favoring net vascular smooth muscle (VSM) relaxation. Alternatively, VHR may be caused by intrinsic VSM desensitization of contraction resulting from prior exposure to high levels of stimuli that temporarily adjusts cell signaling systems. Net relaxation, but not desensitization, would be expected to resolve rapidly in an artery segment removed from the in vivo shock environment and examined in vitro in a fresh solution. Our aim was to 1) induce shock in rabbits and apply an in vitro mechanical analysis on muscular arteries isolated pre- and postshock to determine whether VHR involves intrinsic VSM desensitization, and 2) identify whether net VSM relaxation induced by nitric oxide and cyclic nucleotide-dependent protein kinase activation in vitro can be sustained for some time after relaxant stimulus washout. The potencies of phenylephrine- and histamine-induced contractions in in vitro epigastric artery removed from rabbits posthemorrhage were decreased by ∼0.3 log units compared with the control contralateral epigastric artery removed prehemorrhage. Moreover, a decrease in KCl-induced tonic, relative to phasic, tension of in vitro mesenteric artery correlated with the degree of shock severity as assessed by rates of lactate and K(+) accumulation. VSM desensitization was also caused by tyramine in vivo and PE in vitro, but not by relaxant agents in vitro. Together, these results support the hypothesis that VHR during hemorrhagic decompensation involves contractile stimulus-induced long-lasting, intrinsic VSM desensitization.

Failure of Bay K 8644 to induce RhoA kinase-dependent calcium sensitization in rabbit blood vessels.

BACKGROUND AND PURPOSE: RhoA kinase (ROCK) participates in K(+) depolarization (KCl)-induced Ca(2+) sensitization of contraction. Whether constitutive, depolarization- or Ca(2+)-activated ROCK plays the major role in this signalling system remains to be determined. Here, we determined whether Bay K 8644, a dihydropyridine that promotes Ca(2+) channel clusters to operate in a persistent Ca(2+) influx mode, could cause ROCK-dependent Ca(2+) sensitization. EXPERIMENTAL APPROACH: Renal and femoral artery rings from New Zealand white rabbits were contracted with Bay K 8644. Tissues were frozen and processed to measure active RhoA and ROCK substrate (myosin phosphatase targeting subunit, MYPT1) and myosin light chain (MLC) phosphorylation, or loaded with fura-2 to measure intracellular free Ca(2+) ([Ca(2+)](i)). Effects of selective inhibitors of contraction were assessed in resting (basal) tissues and those contracted with Bay K 8644. KEY RESULTS: Bay K 8644 produced strong increases in [Ca(2+)](i), MLC phosphorylation and tension, but not in MYPT1 phosphorylation. ROCK inhibition by H-1152 abolished basal MYPT1-pT853, diminished basal MLC phosphorylation and inhibited Bay K 8644-induced increases in MLC phosphorylation and tension. MLC kinase inhibition by wortmannin abolished Bay K 8644-induced contraction and increase in MLC phosphorylation but did not inhibit basal MYPT1-pT853. H-1152 and wortmannin had no effect on MYPT1-pT696, but 1 microM staurosporine inhibited basal MYPT1-pT853, MYPT1-pT696 and MLC phosphorylation. CONCLUSIONS AND IMPLICATIONS: These data suggest that the constitutive activities of ROCK and a staurosporine-sensitive kinase regulate basal phosphorylation of MYPT1, which participates along with activation of MLC kinase in determining the strength of contraction induced by the Ca(2+) agonist, Bay K 8644.

Dependency of detrusor contractions on calcium sensitization and calcium entry through LOE-908-sensitive channels.

1. The subcellular mechanisms regulating stimulus-contraction coupling in detrusor remain to be determined. We used Ca(2+)-free solutions, Ca(2+) channel blockers, cyclopiazonic acid (CPA), and RhoA kinase (ROK) inhibitors to test the hypothesis that Ca(2+) influx and Ca(2+) sensitization play primary roles. 2. In rabbit detrusor, peak bethanechol (BE)-induced force was inhibited 90% by incubation for 3 min in a Ca(2+)-free solution. By comparison, a 20 min incubation of rabbit femoral artery in a Ca(2+)-free solution reduced receptor-induced force by only 5%. 3. In detrusor, inhibition of sarcoplasmic reticular (SR) Ca(2+) release by 2APB, or depletion of SR Ca(2+) by CPA, inhibited BE-induced force by only 27%. The CPA-insensitive force was abolished by LaCl3. By comparison, 2APB inhibited receptor-induced force in rabbit femoral artery by 71%. 4. In the presence of the non-selective cation channel (NSCC) inhibitor, LOE-908, BE did not produce an increase in [Ca(2+)]i but did produce weak increases in myosin phosphorylation and force. 5. Inhibitors of ROK-induced Ca(2+) sensitization, HA-1077 and Y-27632, inhibited BE-induced force by approximately 50%, and in combination with LOE-908, nearly abolished force. 6. These data suggest that two principal muscarinic receptor-stimulated detrusor contractile mechanisms include NSCC activation, that elevates [Ca(2+)]i and ROK activation, that sensitizes cross bridges to Ca(2+).