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+).

Regulation of ERK phosphorylation in differentiated arterial muscle of rabbits.

Extracellular signal-regulated kinases (ERK) and mitogen-activated protein (MAP) kinases participate in cell signaling, regulating cell growth. In differentiated cells, the role ERK plays is less well known. This study quantified the degree of basal and stimulated ERK phosphorylation and contraction in freshly isolated arteries. The level of basal ERK phosphorylation was identical in preloaded and slack arteries, was greater in media than in the whole artery, and was reduced by the MAP or ERK kinase (MEK) inhibitor PD-98059. Chemical denudation using 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one did not elevate basal ERK phosphorylation. PD-98059 reduced maximum phenylephrine (PE)-stimulated ERK phosphorylation but not force. Pervanadate elevated ERK phosphorylation without causing contraction. Contractions produced by PE and relaxations produced by PE washout preceded the ERK phosphorylation. K(+) depolarization, muscle stretch, and angiotensin II elevated ERK phosphorylation transiently, whereas PE maintained ERK phosphorylation for 30 min. The alpha(1A)-adrenergic receptor antagonist WB-4101 reduced PE-stimulated force by 70% and abolished PE-induced ERK phosphorylation. Afterloaded and zero-load contractions produced by K(+) depolarization displayed identical increases in ERK phosphorylation. These data indicate that ERK was active basally in the differentiated artery but regulated by the endothelium and that ERK phosphorylation was not load dependent. A strong correlation between PE-induced force and ERK phosphorylation supports the hypothesis that ERK activation may reflect a signal "notifying" the cell of the degree of alpha(1)-adrenergic receptor-induced contraction.

Isolated resistance artery preparation.

The blood vessels that contribute most to precapillary resistance are known as resistance arteries, consisting of arterioles and small arteries with diameters of less than 500 µm (1). These vessels regulate the vascular resistance, and thus the blood supply, through the adjustment of their lumen diameter, which is accomplished by modulation of the level of tone in the vascular smooth muscle cells. The smooth muscle and endothelial cells in the blood vessel wall are sensitive to a great diversity of stimuli including distending pressure, shear stress, neurohumoral factors, and metabolites. All of these different signals are sensed, integrated, and eventually lead to a response.

Temporal aspects of Ca(2+) and myosin phosphorylation during myogenic and norepinephrine-induced arteriolar constriction.

Previous studies demonstrated that maintenance of steady-state myogenic tone requires Ca(2+)-dependent myosin phosphorylation. The present studies furthered these observations by examining temporal relationships among Ca(2+), myosin phosphorylation and vessel diameter during acute increases in intraluminal pressure and norepinephrine stimulation. Rat cremaster muscle arterioles were cannulated and loaded with the Ca(2+)-sensitive indicator fura-2. The extent of myosin phosphorylation was measured using two-dimensional gel electrophoresis. Acute increases in intraluminal pressure caused a biphasic increase in intracellular Ca(2+) ([Ca(2+)](i)), characterized by a transient peak followed by a decline to a steady-state level which remained significantly higher than control values. Peak [Ca(2+)](i) was significantly related to vessel distension and increased with the change in wall tension. Increased intraluminal pressure resulted in a monophasic increase in myosin phosphorylation that was significantly correlated with instantaneous wall tension. In general, norepinephrine induced larger [Ca(2+)](i) transients and a biphasic myosin phosphorylation pattern. The results demonstrate: (a) major roles for Ca(2+) and myosin phosphorylation in arteriolar myogenic and norepinephrine-induced responses; (b) that changes in Ca(2+) and phosphorylation during a myogenic response are related to changes in wall tension, and (c) differences in Ca(2+) and phosphorylation patterns between the two modes of contraction reflect possible differences in underlying signaling mechanisms. The data further emphasize that spontaneous arteriolar tone represents a state of maintained smooth muscle activation that requires increases in [Ca(2+)](i) and myosin light-chain phosphorylation.

Dependence of Ca(2+) sensitivity of arterial contractions on history of receptor activation.

Stimulation of receptors causing arterial contraction may also cause attenuation of cell responsiveness to stimuli. This study tested the hypothesis that attenuation of receptor-induced contractions involves Ca(2+) desensitization. Renal artery rings were pretreated with 10 microM phenylephrine (PE), relaxed with PE washout (plus phentolamine), and then activated by histamine (HA). Pretreatment for 30 min resulted in a rightward shift in the concentration-contraction curve to HA by approximately 1/2 log without a reduction in the slope or maximum response. For example, control and PE-pretreated tissues responded to 0.56 microM HA with strong (0.95 F/F(o)) and weak (0.16 F/F(o)) contractions, respectively, where F/F(o) represents contractile force. This reduced reactivity was completely reversed within 90 min. In fura-loaded tissues, PE pretreatment caused less of a rightward shift in the HA concentration-intracellular free Ca(2+) concentration ([Ca(2+)](i)) curve than in the HA concentration-contraction curve. A dissociation between force and [Ca(2+)](i) was also produced when KCl was used instead of HA. These data suggest that the reduced reactivity produced by PE pretreatment involved, in part, a reduction in the ability of HA to increase the Ca(2+) sensitivity of contractions. These data support the hypothesis that the degree of stimulus-induced Ca(2+) sensitization of contractions is dependent on the history of receptor activation.

Rapid effects of estrogen and progesterone on tone and spontaneous rhythmic contractions of the rabbit bladder.

Previous studies indicate that bladder instability in man may be associated with increased spontaneous rhythmic contractile activity. Ca(2+) influx plays a central role in smooth muscle contractions, and recent evidence suggests that steroid hormones rapidly affect Ca(2+) influx. Therefore we tested the hypothesis that estrogen and progesterone modulates spontaneous rhythmic detrusor contractions. Tissues were secured to isometric force (F) transducers in tissue baths and length-adjusted until K(+)-depolarization produced maximum contractions (F(o)). Spontaneous rhythmic contractions (SRC) were sampled before and immediately after addition of estradiol or progesterone (10(-5) M) to tissue baths. The average frequency and amplitude of SRC were, respectively, 0.156 Hz and 0.053 F/F(o) (n = 24). Estradiol caused an immediate reduction in SRC, such that by 10 min, tone, frequency and amplitude were each reduced by, respectively, 36%, 46% and 47% (n = 7, P < 0.05). However, progesterone caused an immediate weak contraction, and at steady state (10 min), progesterone increased frequency of SRC by 152% but decreased SRC amplitude by 50% (n = 10, P < 0.05). Novel therapies using unique steroids that do not interact with genomic receptors may potentially reduce bladder smooth muscle activity, thereby reducing detrusor instability.

Differential effects of sex hormones and phytoestrogens on peak and steady state contractions in isolated rabbit detrusor.

PURPOSE: Recent evidence suggests that sex steroids may produce rapid inhibition of voltage operated Ca2+ channels (VOCCs). Detrusor smooth muscle is highly dependent upon Ca2+ influx for receptor-activated contractions. Thus, we examined the relative effectiveness of a select group of sex steroids and dietary phytoestrogens to relax detrusor contracted with the muscarinic receptor agonist, bethanechol (BE) and the purinergic P2X receptor agonist, alpha,beta-methylene ATP (alpha,beta-MeATP). MATERIALS AND METHODS: Isolated strips of rabbit detrusor were secured to isometric force transducers in a tissue bath and length-adjusted until maximum contractions were achieved. Peak (P) contractile responses were recorded for alpha,beta-MeATP (P(ATP)) and BE (P(BE)) and steady-state (SS) responses were recorded for BE (SS(BE)) in the presence and absence of selected sex steroids and phytoestrogens (10 microM, unless indicated). RESULTS: The L-type VOCC inhibitor, nifedipine (1 to 10 microM), completely inhibited P(ATP) but reduced SS(BE) by approximately 50%, whereas the VOCC and non-VOCC inhibitor, SKF 96365, inhibited SS(BE) by approximately 95%, suggesting that P(ATP) was entirely dependent on L-type VOCCs, but (BE)-induced contractions depended also on activation of non-VOCCs. 17Beta-estradiol (estradiol) and progesterone inhibited P(ATP) by approximately 60% and 20%, respectively, and 32 microM estradiol and ethinyl estradiol inhibited SS(BE) by approximately 80 and 95%, respectively. Inhibition by estradiol was potentiated, rather than blocked, by the nuclear estrogen receptor antagonist, tamoxifen. Moreover, tamoxifen alone nearly completely relaxed SS(BE). The inactive metabolite of estradiol, 17alpha-estradiol, inhibited both P(ATP) and P(BE) by approximately 40%. Testosterone had no effect on P(ATP) and P(BE). The phytoestrogen and tyrosine kinase inhibitor, genistein, inhibited SS(BE) by 44%, whereas daidzein, a phytoestrogen without tyrosine kinase inhibitory activity, produced only a 7% inhibition. None of the phytoestrogens examined inhibited P(BE), whereas all inhibited P(ATP) by approximately 20 to 35%. A comparison of inhibition of (BE) and alpha,beta-MeATP-induced contractions by selected estrogen isomers showed some distinct differences. For example, estrone did not inhibit P(BE) or SS(BE), but inhibited P(ATP) by approximately 20%, whereas DES inhibited SS(BE) by nearly 90%, but P(ATP) by a lesser degree (approximately 70%). CONCLUSIONS: Our data support the hypothesis that 17beta-estradiol, ethinyl estradiol, DES, tamoxifen and genistein may relax detrusor contractions by inhibition of both VOCCs and non-VOCCs. Moreover, our data show that genistein, a dietary phytoestrogen with tyrosine kinase inhibitory activity, selectively reduced alpha,beta-MeATP-induced peak and BE-induced steady-state contractions, sparing the maximum response to BE. Lastly, the inactive isomer, 17alpha-estradiol, inhibited both BE- and alpha,beta-MeATP-induced contractions. These data suggest that certain dietary phytoestrogens (for example, genistein) or sex steroids, especially those with weak activity at the nuclear steroid site (for example, 17alpha-estradiol), or tamoxifen may prove therapeutically useful in treating overactive bladder caused by elevated muscarinic and purinergic receptor activation.

Bethanechol activates a post-receptor negative feedback mechanism in rabbit urinary bladder smooth muscle.

PURPOSE: Recent studies using vascular and gut smooth muscles indicate that contractile receptor agonists may activate post-receptor down-regulatory mechanisms causing a temporary reduction in the strength of subsequent contractions. Our data indicate a similar mechanism exists in detrusor smooth muscle of the urinary bladder. MATERIALS AND METHODS: Each isolated strip of female rabbit detrusor was placed in a tissue bath, secured to an isometric force transducer, and length-adjusted until depolarization with 110 mM KCl produced a maximum contraction (S0). Subsequent contractions were normalized to S0 (S/S0) or to a first stimulus with 30 mM KCl or caffeine (S/S1). Tissues were pretreated with the muscarinic receptor agonist, bethanechol (BE), then stimulated with KCl, caffeine, or Bay k 8644 to identify potential post-receptor down-regulation. RESULTS: Contractions induced by 30 mM KCl had three phases labeled fast peak (FP), slow peak (SP) and steady-state (SS). In tissues exposed for 30 min. to a maximum BE concentration then washed for 5 min., the KCl-induced FP and SP, but not SS, responses were reduced by approximately 40%. Smaller reductions in peak KCl-induced contractions occurred in tissues pretreated for a shorter duration or with a 100-fold lower BE concentration. This down-regulation induced by bethanechol pretreatment was reversible, lasting approximately 1-2 h. Not only were KCl-induced contractions reduced by BE pretreatment, but also those produced by the intracellular Ca(2+)-mobilizer, caffeine, and the L-type Ca2+ channel agonist, Bay k 8644. CONCLUSIONS: Pretreatment of isolated strips of rabbit detrusor with a muscarinic receptor agonist produced short-term down-regulation of KCl-induced peak contractions that may have involved inhibition of both influx of extracellular Ca2+ and release of intracellular Ca2+. Reductions in the degree of this novel modulatory response during disease conditions and aging could enhance contractile activity, possibly causing detrusor instability.