Sympathetic nerves and the endothelium influence the vasoconstrictor effect of low concentrations of ouabain in pressurized small arteries.

We hypothesized that in salt-dependent forms of hypertension, endogenous ouabain acts on arterial smooth muscle to cause enhanced vasoconstriction. Here, we tested for the involvement of the arterial endothelium and perivascular sympathetic nerve terminals in ouabain-induced vasoconstriction. Segments of rat mesenteric or renal interlobar arteries were pressurized to 70 mmHg at 37 degrees C and exposed to ouabain (10(-11)-10(-7) M). Removal of the endothelium enhanced ouabain-induced vasoconstriction by as much as twofold (at an ouabain concentration of 10(-9) M). A component of the ouabain-induced vasoconstriction is due to the enhanced spontaneous release of norepinephrine (NE) from nerve terminals in the arterial wall. The alpha(1)-adrenoceptor blocker prazosin (10(-6) M) decreased ouabain-induced vasoconstrictions by as much as 50%. However, neither the contraction induced by sympathetic nerve activity (SNA) nor the NE release evoked by SNA (measured directly by carbon fiber amperometry) was increased by ouabain (<10(-7) M). Nevertheless, the converse case was true: after brief bursts of SNA, vasoconstrictor responses to ouabain were transiently increased (1.75-fold). This effect may be mediated by neuropeptide Y and Y(1) receptors on smooth muscle. In arteries lacking the endothelium and exposed to prazosin, ouabain (10(-11) M and greater) caused vasoconstriction, indicating a direct effect of very "low" concentrations of ouabain on arterial smooth muscle. In conclusion, in intact arteries, the endothelium opposes ouabain (10(-11)-10(-7)M)-induced vasoconstriction, which is caused by both enhanced spontaneous NE release and direct effects on smooth muscle. Ouabain (<10(-7)M) does not enhance SNA-mediated contractions, but SNA enhances ouabain-induced contractions. The effects of endogenous ouabain may be accentuated in forms of hypertension that involve sympathetic nerve hyperactivity and/or endothelial dysfunction.

Vascular reactivity in heart failure: role of myosin light chain phosphatase.

Congestive heart failure (CHF) is a clinical syndrome, which is the result of systolic or diastolic ventricular dysfunction. During CHF, vascular tone is regulated by the interplay of neurohormonal mechanisms and endothelial-dependent factors and is characterized by both central and peripheral vasoconstriction as well as a resistance to nitric oxide (NO)-mediated vasodilatation. At the molecular level, vascular tone depends on the level of regulatory myosin light chain phosphorylation, which is determined by the relative activities of myosin light chain kinase and myosin light chain phosphatase (MLCP). The MLCP is a trimeric enzyme with a catalytic, a 20-kDa and a myosin targeting (MYPT1) subunit. Alternative splicing of a 3' exon produces leucine zipper positive and negative (LZ+/-) MYPT1 isoforms. Expression of a LZ+ MYPT1 has been suggested to be required for NO-mediated smooth muscle relaxation. Thus, we hypothesized that the resistance to NO-mediated vasodilatation in CHF could be attributable to a change in the relative expression of LZ+/- MYPT1 isoforms. To test this hypothesis, left coronary artery ligation was used to induce CHF in rats, and both the dose response relationship of relaxation to 8-Br-cGMP in skinned smooth muscle and the relative expression of LZ+/- MYPT1 isoforms were determined. In control animals, the expression of the LZ+ MYPT1 isoform predominated in both the aorta and iliac artery. In CHF rats, LVEF was reduced to 30+/-5% and there was a significant decrease in both the sensitivity to 8-Br-cGMP and expression of the LZ+ MYPT1 isoform. These results indicate that CHF is associated with a decrease in the relative expression of the LZ+ MYPT1 isoform and the sensitivity to 8-Br-cGMP-mediated smooth muscle relaxation. The data suggest that the resistance to NO-mediated relaxation observed during CHF lies at least in part at the level of the smooth muscle and is a consequence of the decrease in the expression of the LZ+ MYPT1 isoform.

Nonmuscle myosin, force maintenance, and the tonic contractile phenotype in smooth muscle.

Recent studies have demonstrated that nonmuscle (NM) myosin II forms filaments and can generate and maintain force in smooth muscle tissue [Lofgren et al. (2003) J Gen Physiol 121:301-310; Morano et al. (2000) Nat Cell Biol 2:371-375]. To further investigate the mechanical contribution of NM myosin to force maintenance during smooth muscle contraction, we utilized a selective inhibitor of the NM myosin ATPase, blebbistatin [Straight et al. (2003) Science 299:1743-1747]. Force and myosin light chain (MLC(20)) phosphorylation were measured during KCl stimulation of small strips of intact mouse bladder and aorta at 22 degrees C. The bladder strips contracted with a typical phasic force response, characterized by a large, rapid, transient increase in force followed by a decline to a lower, steady-state level. The addition of blebbistatin did not alter the peak force, but decreased force maintenance. KCl depolarization of aortic strips resulted in a tonic contraction; force increased to a sustained steady state. Similar to the bladder tissue, blebbistatin substantially decreased the steady-state force in the aorta. Blebbistatin did not influence the MLC(20) phosphorylation transient in either tissue type. Additionally, blebbistatin did not change the maximum shortening velocity (V (max)) during KCl depolarization of the aorta. Our results also suggest that NMIIA and NMIIB isoforms are differentially expressed. The expression of NMIIA is more prominent in the bladder, while NMIIB expression is predominant in the aorta. These results suggest that NM myosin contributes to the mechanism of force maintenance in smooth muscle, and could suggest that the expression of NMIIB is a factor for determining the tonic contractile phenotype.

Captopril prevents myosin light chain phosphatase isoform switching to preserve normal cGMP-mediated vasodilatation.

Congestive heart failure (CHF) is characterized by abnormal vasoconstriction and an impairment in nitric oxide (NO)-mediated vasodilatation. We have previously demonstrated that the decrease in sensitivity to NO lies at least partially at the level of the smooth muscle and is due to a reduction in the relative expression of the leucine zipper positive (LZ(+)) isoform of the myosin targeting subunit (MYPT1) of myosin light chain phosphatase. We hypothesized that since the attenuated vasodilatory response to NO in CHF has been shown to be secondary to an increased activity of the renin-angiotensin system, angiotensin converting enzyme (ACE) inhibition could affect MYPT1 isoform expression. To test this hypothesis, a rat myocardial infarction (MI) model of CHF was used; following left coronary artery ligation, rats were divided into control and captopril-treated groups. A third group of rats was given prazosin for 4 weeks. In the untreated control group, left ventricular function (LVF) was reduced at 2 weeks post-MI and remained at this level. Captopril treatment attenuated the fall in LVF. In the control aorta and iliac artery, the expression of the LZ(+) MYPT1 isoform fell 44-52% between 2 and 4 weeks post-MI, whereas in animals treated with captopril, MYPT1 isoform expression did not change. A decrease in the sensitivity to cGMP-mediated smooth muscle relaxation occurred coincident with the decrease in LZ(+) MYPT1 expression. The change in LZ(+) MYPT1 expression was not due to the decrease in afterload, as prazosin therapy produced an improvement in LVF but did not increase the relative expression of LZ(+) MYPT1 isoform. These data suggest that ACE inhibition, unique from pure afterload reduction, prevents MYPT1 isoform switching, which would preserve normal flow, or NO-mediated vasodilatation.

Effects of h1-calponin ablation on the contractile properties of bladder versus vascular smooth muscle in mice lacking SM-B myosin.

The functional significance of smooth muscle-specific h1-calponin up-regulation in the smooth muscle contractility of SM-B null mice was studied by generating double knockout mice lacking both h1-calponin and SM-B myosin. The double knockout mice appear healthy, reproduce well and do not show any smooth muscle pathology. Loss of h1-calponin in the SM-B null mice bladder resulted in increased maximal shortening velocity (V(max)) and steady-state force generation. The force dilatation pressure, which was decreased in the SM-B null mesenteric vessels, was restored to wild-type levels in the double knockout vessels. In contrast, the half-time to maximal constriction was significantly increased in the double knockout vessels similar to that of SM-B null mice and indicating decreased shortening velocity in the double knockout vessels. Biochemical analyses showed that there is a significant reduction in smooth muscle alpha-actin levels, whereas h-caldesmon levels are increased in the double knockout bladder and mesenteric vessels, suggesting that these changes may also partly contribute to the altered contractile function. Taken together, our studies suggest that up-regulation of h1-calponin in the SM-B null mice may be necessary to maintain a reduced level of cross-bridge cycling over time in the absence of SM-B myosin and play an important role in regulating the smooth muscle contraction.

Force maintenance in smooth muscle: analysis using sinusoidal perturbations.

The "latch state" or force maintenance may be due to the emergence of a distinct set of dephosphorylated, slowly cycling "latch" cross-bridges, slowing of the overall cross-bridge cycling rate, or a non-cross-bridge contribution. This was investigated by sinusoidally oscillating strips of intact rabbit portal vein or aorta. Tissue strips were activated with KCl depolarization, resulting in a sustained increase of MLC(20) phosphorylation or 10 microM phenylephrine, resulting in a transient increase in MLC(20) phosphorylation. Stiffness was calculated from the force response to a small, sine-wave oscillation in muscle length (1-100 Hz). The results produced a 3-dimensional plot of stiffness versus the frequency of oscillation (Hz) versus time (s), or stiffness distribution profile. During KCl depolarization, the stiffness distribution profile displayed a shift toward lower frequencies, suggesting a general slowing in the overall cross-bridge cycling rate during force maintenance. On the other hand, phenylephrine stimulation did not display a significant change in the stiffness distribution profile, suggesting that the overall cross-bridge cycling rate did not significantly change during force maintenance.