Role of protein kinase C in myogenic calcium-contraction coupling of rat cannulated mesenteric small arteries.

1. The present study was designed to determine the role of protein kinase C (PKC) in the myogenic response of small arteries. In particular, we tested whether inhibition of PKC reverses the previously found pressure-induced elevation of contractile element calcium sensitivity. 2. Rat mesenteric small arteries were cannulated and pressurized. The internal diameter was continuously monitored with a video camera and intracellular calcium levels were measured by means of fura-2. Myogenic responses were observed when the pressure was raised stepwise from 20 to 60 and then to 100 mmHg in physiological saline solution and during application of phenylephrine (0.1 or 1 micromol/L) or potassium (36 mmol/L). 3. The PKC inhibitors H-7 (20 micromol/L), staurosporine (100 nmol/L) and calphostin C (10 nmol/L) all completely abolished the myogenic response. Whereas staurosporine caused an ongoing reduction in intracellular calcium, pressure-induced calcium transients were not affected by either H-7 or calphostin C. In particular, the slope of the wall tension-calcium relationship remained similar in the presence of both H-7 and calphostin C, despite an upward shift of this relationship to higher calcium levels in the case of calphostin C. 4. These results show that activity of PKC isoform(s) is essential for myogenic calcium-contraction coupling.

Src tyrosine kinases and extracellular signal-regulated kinase 1/2 mitogen-activated protein kinases mediate pressure-induced c-fos expression in cannulated rat mesenteric small arteries.

Chronic hypertension is associated with remodeling of small arteries. There is evidence that the high pressure itself may cause these structural changes, but the responsible mechanisms are not clearly defined. Previously we showed that pressure-induced c-fos expression in intact cannulated rat mesenteric small arteries was inhibited by genistein, a general tyrosine kinase inhibitor. The purpose of this study was to further unravel the underlying signal transduction mechanisms, and we particularly tested the involvement of src tyrosine kinases and extracellular signal-regulated kinase (ERK). Rat mesenteric small arteries were cannulated in a dual-vessel chamber. After a 60-minute equilibration period, the pressure in 1 artery was increased to 140 mm Hg, while the other artery remained at 90 mm Hg. Semiquantitative reverse transcriptase-polymerase chain reaction was used to determine c-fos expression, and Western blotting was used to examine levels of ERK phosphorylation. The involvement of src and ERK was tested with the inhibitors herbimycin A (1 micromol/L), PP1 (10 micromol/L), PP2 (10 micromol/L), and PD98059 (30 micromol/L). One-hour exposure to 140 mm Hg increased the c-fos/cyclophilin ratio 3.6-fold, from 0.29+/-0.07 to 1.06+/-0.25. All the tested inhibitors suppressed the pressure-induced increase of c-fos expression. A 5-minute exposure period to 140 mm Hg increased ERK phosphorylation, and this was abolished in the presence of PP1. The results suggest that pressure-induced c-fos expression in intact cannulated rat mesenteric small arteries may be mediated, at least in part, by src tyrosine kinases and ERK.

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.

Myogenic activation and calcium sensitivity of cannulated rat mesenteric small arteries.

Pressure-induced activation of vascular smooth muscle may involve electromechanical as well as nonelectromechanical coupling mechanisms. We compared calcium-tone relations of cannulated rat mesenteric small arteries during pressure-induced activation, depolarization (16 to 46 mmol/L K+), and alpha1-adrenergic stimulation (1 micromol/L phenylephrine). The intracellular calcium concentration was expressed as the fura-2 ratio, normalized to the maximal and minimal ratios. In order to compare activation levels at various pressures, tone was expressed as the ratio of active wall tension to the maximal active tension. The passive and maximal active pressure-diameter relations needed for the calculation of tone were determined in a separate set of experiments, using isometric loading of cannulated vessels. Pressure steps from 20 to 60 and then to 100 mm Hg caused a modest rise of calcium. Nifedipine (1 micromol/L) blocked both the calcium rise and the resulting myogenic responses. Electromechanical coupling could not fully account for the myogenic response: the calcium sensitivity, defined as the slope of the calcium-tone relation, was five times higher during pressure-induced activation compared with potassium stimulation and twice as high as the sensitivity during alpha1-adrenergic stimulation. We therefore conclude that the myogenic response involves a small but necessary rise in calcium due to influx through L-type calcium channels, as well as a nonelectromechanical coupling mechanism that greatly enhances the calcium sensitivity of the contractile machinery.

KCa-channel blockade prevents sustained pressure-induced depolarization in rat mesenteric small arteries.

In small blood vessels, elevation of transmural pressure induces myogenic constrictions and smooth muscle depolarization. The role of calcium-activated K+ channels (KCa channels) in these responses was examined in cannulated rat mesenteric small arteries. Inner and outer diameter were continuously monitored with a video technique. Smooth muscle membrane potential was recorded simultaneously using microelectrodes. To test for myogenic responsiveness, the transmural pressure was changed stepwise in the range between 10 and 120 mmHg. Pressure elevation induced moderate myogenic responses and significant depolarization, from -54.5 +/- 0.4 (SE) mV (n = 56) at 10 mmHg to -47.3 +/- 1.8 mV (n = 12) at 120 mmHg. Norepinephrine (NE, 0.67 and 10 microM) induced constriction and vasomotion, augmented myogenic responsiveness, and shifted the pressure-membrane potential relation to more depolarized values. Blockade of the Kca channels with charybdotoxin (ChTX) suppressed the responsiveness to pressure. In the absence of ChTX, with 0.67 microM NE, pressure elevation from 10 to 120 mmHg induced depolarization from -46.9 +/- 1.0 (n = 16) to -35.8 +/- 0.7 (n = 12) mV, whereas because of the myogenic response, the diameter increased only by 7%. In the presence of ChTX, with 0.3 microM NE, pressure changed the membrane potential from -41.0 +/- 1.1 (n = 12) to -37.8 +/- 0.7 mV (n = 4), which was not significant, and the diameter increased by 28%. These results demonstrate that blockade of KCa channels reduces responsiveness to pressure elevation. This suggests that pressure may induce depolarization and concomitant myogenic responsiveness by closure of KCa channels.

Noradrenaline-induced depolarization is smaller in isobaric compared to isometric preparations of rat mesenteric small arteries.

The hypothesis was tested that wall tension can influence the membrane potential response to noradrenaline (NA) using isometric and isobaric vessel preparations of rat mesenteric small arteries. The resting membrane potential was significantly less negative in the isobaric (-49.7+/-0.5 mV, S.E.M., n=12 vessels) compared to the isometric preparation (-56.1+/-0.7 mV, n=10), although there was no difference in wall tension. The depolarization induced by 10(-5) M NA was 2.6-fold smaller in the isobaric preparation, where wall tension decreased, compared to the isometric preparation, where wall tension increased. Since wall tension decreases under isobaric conditions, but increases under isometric conditions, the latter finding can be explained by assuming that part of the NA-induced membrane potential change is wall tension dependent.

Voltage-operated calcium channels are essential for the myogenic responsiveness of cannulated rat mesenteric small arteries.

The role of L-type voltage-operated Ca2+ channels (VOCs) in myogenic responsiveness was studied in cannulated rat mesenteric small arteries [mean diameter at 100 mm Hg and full dilation was 329 +/- 9 (SE) micrometer]. Twenty-six arteries were cannulated and pressurized. The luminal cross-sectional area of these vessels was monitored continuously. To test for myogenic responsiveness, pressure was raised stepwise from 20 to 60 and from 60 to 100 mm Hg. Pressure elevation enhanced the vascular tone, reflecting spontaneous myogenic responsiveness. Nifedipine (1 and 10 microM) suppressed spontaneous myogenic responses. The alpha1-adrenoceptor agonist phenylephrine (1 and 10 microM), when administered at 20 mm Hg, elicited constriction and vasomotion, and potentiated myogenic constriction to subsequent pressure elevation. Nifedipine (1 and 10 microM) also suppressed phenylephrine-potentiated myogenic responsiveness. Stimulation of VOCs with BAY K 8644 (10-300 nM) had no effect at 20 mm Hg, but augmented myogenic responsiveness. K+ (16-46 mM) caused concentration-dependent constrictions when administered at 20 mm Hg, and potentiated myogenic responsiveness when the pressure was raised from 20 to 60 mm Hg. Thus, any intervention that blocked the VOCs also blocked myogenic responses. Therefore, we conclude that VOCs are essential for the myogenic responsiveness of cannulated rat mesenteric small arteries.

The effects of pentobarbital and benzodiazepines on GABA-responses in the periphery and spinal cord in vitro.

Pentobarbital and benzodiazepines were compared in their interaction with the gamma-aminobutyric acid (GABA) antagonists picrotoxin and bicuculline on GABAA receptor-mediated events. On excised vagal nerves and dorsal roots pentobarbital, in contrast to the benzodiazepines diazepam, lorazepam and flurazepam, was able to enhance GABA-induced depolarizations recorded in the presence of picrotoxin or bicuculline. On hemicord preparations picrotoxin simultaneously depressed the electrically evoked dorsal root-dorsal root potential and enhanced the dorsal root-ventral root potential. Pentobarbital overcame the effects of picrotoxin, whereas diazepam and midazolam were without effect. These results may be explained by the suggestion that the GABA receptors in these test systems are not tightly associated with the benzodiazepine receptor activated by diazepam, lorazepam, midazolam and flurazepam, and correspond to the recently described GABAA2 subdivision of GABA receptors.