It is generally assumed that smooth muscle contraction is dependent on changes in intracellular Ca2+ concentration ([Ca2+]i); however, we have previously reported that alpha-agonist-induced contraction of aorta smooth muscle cells can occur in the absence of changes in [Ca2+]i [Collins, E. M., M. P. Walsh, and K. G. Morgan. Am. J. Physiol. 262 (Heart Circ. Physiol. 31): H754-H762, 1992]. The mechanism of this [Ca2+]i-independent contraction is controversial. We have now identified the Ca(2+)-independent protein kinase C (PKC) isoforms epsilon and zeta in ferret aorta and have used digital imaging microscopy to determine their subcellular distribution. At rest, epsilon-PKC is diffusely distributed in the cytosol, whereas zeta-PKC is concentrated in the perinuclear region; both isoforms are excluded from the nuclear space. Agonist stimulation causes a [Ca2+]i-independent translocation of epsilon-PKC to the surface membrane and of zeta-PKC to the intranuclear compartment. In comparison, ferret portal vein cells, which display a totally Ca(2+)-dependent agonist contraction, are lacking in epsilon-PKC but display perinuclear zeta-PKC, which translocates intranuclearly on activation. Thus the Ca(2+)-independent vascular contraction appears to be associated with plasmalemmal translocation of epsilon-PKC; in contrast, the intranuclear translocation of zeta-PKC may function in control of gene expression.

It remains presently unknown whether vascular reactivity is impaired and whether maladaptive cardiac remodeling occurs before the onset of overt obesity and in the absence of hyperlipidemia. Normal female rats were fed a high-fat diet for 8 weeks and were associated with a modest nonsignificant increase of body weight (standard diet, 300 Ϯ 10, versus high-fat diet, 329 Ϯ 14 g) and a normal plasma lipid profile. In rats fed a high-fat diet, systolic (171 Ϯ 7 mm Hg) and diastolic blood pressures (109 Ϯ 3) were increased compared to a standard diet (systolic blood pressure, 134 Ϯ 8; diastolic blood pressure, 96 Ϯ 5 mm Hg), and acetylcholine-dependent relaxation of isolated aortic rings (high-fat diet, 22 Ϯ 5%, versus standard diet, 53 Ϯ 8%) was significantly reduced. Furthermore, perivascular fibrosis was detected in the heart of rats fed a high-fat diet. The exogenous addition of resveratrol (trans-3,5,4Ј-trihydroxystilbene) (0.1 M) to aortic rings isolated from rats fed a high-fat diet restored acetylcholine-mediated relaxation (47 Ϯ 9%). The administration of resveratrol (20 mg/kg/day for 8 weeks) to rats fed a high-fat diet prevented the increase in blood pressure and preserved acetylcholine-dependent relaxation of isolated aortic rings. However, resveratrol therapy failed to attenuate the perivascular fibrotic response. These data have demonstrated that a high-fat diet fed to normal female rats can elicit a hypertensive response and induce perivascular fibrosis before the development of overt obesity and in the absence of hyperlipidemia. Resveratrol therapy can prevent the hypertensive response in female rats fed a high-fat diet but is without effect on the progression of perivascular fibrosis.

Because of inherent difficulties in maintaining physiological conditions in biochemical assays, the intracellular free Ca2+ concentration ([Ca2+]i) required for activation of protein kinase C (PKC) in intact cells remains unclear. In the present study, [Ca2+]i was measured in freshly isolated vascular smooth muscle cells loaded with fura 2 while, in parallel, the distribution of the Ca(2+)-dependent alpha-PKC isoform was monitored using digital imaging microscopy. The [Ca2+]i alpha-PKC translocation threshold was determined by changing extracellular free Ca2+ concentration in steps while monitoring [Ca2+]i. In the absence of agonists, increasing [Ca2+]i caused < 25% of maximal translocation. In the presence of phenylephrine, maximum translocation occurred at [Ca2+]i > or = 198 nM. Phenylephrine augmented translocation of alpha-PKC primarily by increasing the slope of the [Ca2+]i-PKC translocation relationship. These results indicate that the [Ca2+]i threshold of alpha-PKC translocation in situ is less than that reported in most in vitro assays and are consistent with an effect of agonist-induced generation of other second messengers that cause cooperative interactions leading to translocation.

Changes in physiological variables during a 60-min continuous test at maximal lactate steady state (MLSS) were studied using highly conditioned cyclists (1 female and 9 males, aged 28.3 +/- 8.1 years). To determine power at MLSS, we tested at 8-min increments and interpolated the power corresponding to a blood lactate value of 4 mmol/L. During the subsequent 60-min exercise at MLSS, we observed a sequential increase of physiological parameters, in contrast to stable blood lactate. Heart rate drifted upward from beginning to end of exercise. This became statistically significant after 30 min. From 10-60 min of exercise, a change of +12.6 +/- 3.2 bpm was noted. Significant drift was seen after 30 min for the respiratory exchange ratio, after 40 min for the rate of perceived exertion using the Borg scale, and after 50 min for % VO(2)max/kg and minute ventilation. This slow component of VO(2)max may be the result of higher recruitment of type II fibers.