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Effects of genistein, a tyrosine kinase inhibitor, on increases in [Ca 2+ ] i and protein tyrosine phosphorylation induced by 20 n M [arginine 8 ]vasopressin (AVP) were studied in A7r5 aortic smooth muscle cells. In fura-2-loaded cells, AVP induced a rapid (0.5–2 min) transient increase in [Ca 2+ ] i that was followed by a smaller sustained increase in [Ca 2+ ] i . In 66% of the cells, the transient response involved both influx of extracellular Ca 2+ and release of intracellular Ca 2+ : influx accounted for 60% of the response, and release accounted for 40%. However, in 34% of the cells, the relative contribution of influx and release during the transient could not be assessed. In all cells, the smaller sustained response was entirely dependent on extracellular Ca 2+ . Genistein (148 μ M ) always blocked the transient and sustained components of the Ca 2+ response showing that both influx and release were genistein-sensitive. Isobestic fluorescence analysis, in medium containing 0.5 m M Mn 2+ in place of Ca 2+ , showed that the influx pathway was selective because it did not conduct Mn 2+ . It also confirmed that Ca 2+ release was blocked by genistein. In contrast, 105 μ M lavendustin A, a different tyrosine kinase inhibitor, suppressed the transient by only 30%. Another inhibitor, tyrphostin 47 (80 μ M ), did not alter the transient or sustained components of the Ca 2+ response. No AVP-induced increases in tyrosine phosphorylation were detected unless special procedures were used. When cells were preincubated in 10 m M vanadate, a tyrosine phosphatase inhibitor, AVP induced a transient increase in tyrosine phosphorylation (5–60 s). The time course for AVP-induced phosphorylation was similar to that for increases in [Ca 2+ ] i . Vanadate alone increased tyrosine phosphorylation and induced a slow small increase in [Ca 2+ ] i that was dependent on extracellular Ca 2+ . Genistein blocked tyrosine phosphorylation induced by AVP and vanadate, and it blocked the increase in [Ca 2+ ] i induced by vanadate alone. In contrast, lavendustin or tyrphostin unexpectedly enhanced tyrosine phosphorylation induced by vanadate alone and precluded assessment of AVP-induced tyrosine phosphorylation in the presence of vanadate. Levandustin produced time-dependent enhancement of vanadate-induced increase in [Ca 2+ ] i . These results underscored the need for measuring cellular changes in protein tyrosine phosphorylation to assess potential functions of tyrosine kinase activity. Under conditions where changes in phosphorylation could be measured, the results suggested that AVP-activated increases in tyrosine phosphorylation may be coupled to AVP-activated mechanisms that regulate influx of extracellular Ca 2+ and release of intracellular Ca 2+ .

This study was guided by the hypothesis that specific isoforms of protein kinase C may participate in modulating increases in intracellular Ca2+ that are induced by stimulation of vascular smooth muscle cells with vasopressin. Immunoblot analysis revealed that A7r5 vascular smooth muscle cells expressed conventional (alpha), novel (delta and epsilon), and atypical (iota/lambda and mu) isoforms of protein kinase C. Stimulation of fura-2-loaded cells with 20 nM vasopressin induced a rapid transient increase in the intracellular concentration of calcium that was followed by a slowly declining component which was above baseline throughout the period of observation. Cell fractionation studies showed that the calcium response was associated with (a) transient translocation of the alpha and delta isoforms of protein kinase C from the cytosolic fraction to the particulate-membrane fraction, (b) sustained translocation of the epsilon isoform, and (c) no translocation of iota/lambda or mu isoforms. Ratiometric and isobestic fluorescence analysis showed that vasopressin-induced Ca2+ influx and release were markedly inhibited in cells that were preincubated with either 1 microM phorbol 12-myristate 13-acetate, or 10 microM 1, 2 dioctanoyl-sn-glycerol, two structurally different activators of protein kinase C. In contrast, vasopressin-induced increases in intracellular Ca2+ were not significantly altered following preincubation with either 1 microM 4alpha-phorbol or 4alpha-phorbol 12,13-didecanoate, analogs of phorbol 12-myristate 13-acetate that do not activate protein kinase C. Moreover, the inhibitory effects of phorbol 12-myristate 13-acetate were prevented by treatment with 1 microM GF109203X, a potent inhibitor of protein kinase C. Taken together, these results show that direct activation of protein kinase C can negatively modulate vasopressin-induced Ca2+ influx and release in cultured vascular smooth muscle cells. They also show that stimulation with vasopressin induces translocation of specific isoforms of protein kinase C, an observation suggesting that one or more of these isoforms may participate in modulation of vasopressin-induced increases in intracellular Ca2+.

Recent studies showed that serotonin-activated increases in intracellular Ca2+in vascular smooth muscle cells are associated with enhanced protein tyrosine phosphorylation. These responses were blocked by inhibition of tyrosine kinase activity with genistein, suggesting that the increases in Ca2+and tyrosine phosphorylation are functionally coupled. Therefore, we sought to characterize genistein-sensitive Ca2+transport pathways in rat aortic A10 cells loaded with fura-2. In the presence of extracellular Ca2+, serotonin evoked a transient increase in [Ca2+]ithat was followed by a smaller sustained increase. The transient was inhibited 25–40% by L-type Ca2+channel antagonists and inhibited 90–95% by genistein. The sustained response was unaffected by L-channel antagonists and only slightly inhibited by genistein. In the absence of extracellular Ca2+, the transient was reduced by 50%, while the sustained component was virtually abolished. These results suggest that influx and release pathways are major contributors to the transient component, whereas the lower sustained component is largely limited to influx pathways. The influx pathway during the transient probably involves an L-type Ca2+channel that is regulated by tyrosine kinase activity. The pathways that participate in the sustained response are different because they are insensitive to l -channel antagonists and only slightly inhibited by genistein. The transient evoked in Ca2+-free media was blocked by genistein, inhibited by caffeine, and prevented by thapsigargin. Ionomycin-induced release of Ca2+was unaffected by genistein, reduced by caffeine, and essentially eliminated by thapsigargin. Therefore, thapsigargin-mediated suppression of serotonin-activated release probably reflects depletion of Ca2+from the sarcoplasmic reticulum, whereas genistein-mediated suppression probably reflects inhibition of tyrosine kinase linked release. Caffeine-mediated suppression appears to involve both partial depletion of Ca2+and interference with release. Each A10 cell expressed at least two different ryanodine receptors and two different receptors for inositol 1,4,5-trisphosphate.

This review addresses a rapidly growing body of evidence suggesting that enhanced protein tyrosine phosphorylation may be a previously unrecognized mechanism for coupling receptor activation of vascular smooth muscle cells to increases In the intracellular concentration of Ca2+ and contraction. The hypothesis proposes that activation of diverse types of receptors that are not tyrosine kinase promotes stimulation of a cytosolic tyrosine kinase. In turn, the activated kinase induces tyrosine phosphorylation of substrates that are linked to regulatory mechanisms for release of intracellular Ca2+ stored in the sarcoplasmic reticulum and to regulatory mechanisms for influx of extracellular Ca2+. Within this framework, we examine some relevant functional aspects of receptor and nonreceptor tyrosine kinases in different types of cells, the emerging relationships between tyrosine kinase activity and regulation of intracellular Ca2+. We review studies of nonreceptor tyrosine kinase activity in vascular smooth muscle cells suggesting that a physiologically relevant kinase may be the enzyme called pp60. Data that appear to link tyrosine phosphorylation to contraction of smooth muscle are examined, particularly with respect to results obtained with tyrosine kinase inhibitors and measures of changes in tyrosine phosphorylation. Next, we review studies with cultured vascular smooth muscle cells that point to potential coupling between receptor activation, enhanced tyrosine phosphorylation of substrates such as the GTPase activating protein for ras, and the gamma-1 isoform of phospholipase C, and mechanisms controlling Ca2+ influx and release. Emphasis is placed on examining the strengths and weaknesses of different experimental approaches. Lastly, a summary of the data is provided which calls attention to some major issues requiring resolution to permit acceptance or rejection of the underlying hypothesis, and we briefly address some of its possible pathophysiological implications.

A multisubstrate Ca2+ and cyclic nucleotide independent kinase (Mr = 47,000) was purified from bovine aortic smooth muscle. Phosphorylation of glycogen synthase by this enzyme was polycation modulable. Low concentrations of polylysine (0.04-0.16 microM) stimulated phosphorylation 2-7 fold, whereas higher concentrations suppressed phosphorylation. Glycogen synthase converted to its glucose 6-PO4 dependent form following phosphorylation in either the presence (7 mol 32P/mol synthase) or absence (4 mol 32P/mol synthase) of polylysine: extent of conversion correlated to extent of phosphorylation. Seven of 14 potential substrates tested were phosphorylated: kinase activity was greatest for phosvitin followed by casein, the receptor protein from type 2 cAMP-kinase, histone H2b, phosphorylase kinase, glycogen synthase, and myocardial myosin light chains. Phosphorylation of phosvitin or synthase was inhibited by heparin (1/2 maximally by 0.5 microgram/ml without salt and 37 micrograms/ml with 150 mM NaCl). The results suggest that the enzyme may participate in regulating arterial glycogen metabolism and that such regulation may be modulated by polycationic and polyanionic effectors.

An aortic phosphatase which dephosphorylates several proteins including phosphorylase a and the 20-kDa myosin light chains is subject to modulation in vitro by polycationic effectors such as lysine-rich histone-H1 and polylysine. This study was based on the hypothesis that polycationic modulation of expressed enzymic activity involves interactions between the effectors and a regulatory site associated with the polycation-modulated (PCM)-phosphatase. Basal PCM-phosphatase activity expressed against myocardial myosin light chains (MLC, 1258 nmole/min/mg) was about eightfold greater than activity expressed against phosphorylase a (149 nmole/min/mg). However, dephosphorylation of phosphorylase a was stimulated four- to sevenfold by low concentrations of polylysine (Mr = 13,000; 0.01-0.1 microM), whereas MLC phosphatase activity was virtually abolished. Higher concentrations of polylysine inhibited dephosphorylation of either substrate. Interestingly, a heat-stable fraction prepared from the PCM-phosphatase reversed the stimulatory effect of polylysine on phosphorylase phosphatase activity and the inhibitory effect on dephosphorylation of MLC. No reversal of the modulatory effects of polylysine occurred when protein phosphatase inhibitor 1 or inhibitor 2 was substituted for the heat-stable factor derived from the PCM-phosphatase. Sucrose density centrifugation of the enzyme yielded a single peak (Mr = 63,000) exhibiting polycation-modulated activity against phosphorylase a and MLC. Moreover, heating each of the gradient fractions showed the presence of a heat-stable factor which reversed the modulatory effects of polylysine on dephosphorylation of either phosphorylase a or MLC. These results show that a specific heat-stable factor, which differs from both inhibitor 1 and 2, is associated with the PCM-phosphatase. The results suggest that polycationic modulation of expressed PCM-phosphatase activity may involve interactions between the polycationic effector and the enzyme-associated regulatory factor.

Summary Myosin light chain phosphorylation in aortic smooth muscle homogenate reached a maximal level of 0.75 mol phosphate/mol light chain, and then declined. Addition of okadaic acid led to a sustained phosphorylation level of 1.7 mol/mol. In the absence of okadaic acid, phosphorylation was predominantly due to myosin light chain kinase, whereas in the presence of okadaic acid both myosin light chain kinase and protein kinase C were involved in phosphorylation. Okadaic acid inhibited dephosphorylation of the distinct sites in LC phosphorylated by either myosin light chain kinase or protein kinase C, suggesting that it exerts its effect through inhibition of myosin light chain phosphatases present in aortic homogenate.

Several protein phosphatases which we designated PCM-I, PCM-II and PCM-II d were identified in preparations from aortic smooth muscle. A unique feature of these enzymes is that phosphatase activities expressed against different substrates are subject to modulation by polycationic effectors such as polylysine and lyine-rich histone-H 1 . They can be distinguished from each other by virtue of significant quantitative differences regarding (a) relative substrate specificities exhibited against phosphorylated myosin light chains, phosphorylase, and inhibitor-1, (b) apparent molecular weights as determined by sucrose density centrifugation, and (c) differential susceptibility to polylysine-mediated modulation of phosphatase activities. Surprisingly, gel filtration of the very same PCM-phosphatase preparation yields either of two apparently different enzymes: namely PCM-II, or PCM-II d . The enzymes appear similar in that low concentrations of polylysine (0.03–0.13 μ m ) inhibit dephosphorylation of light chains by either enzyme and high concentrations inhibit dephosphorylation of phosphorylase a . However, PCM-II exhibits higher basal phosphatase activity against myosin light chains (480 U/mg) than against phosphorylase a (175 U/mg). In contrast, PCM-II d is more effective in dephosphorylating phosphorylase a (180 U/mg) than in dephosphorylating the light chains (88 U/mg). Moreover, phosphorylase phosphatase activity of PCM-II is biphasically stimulable by low concentrations of polylysine (0.01–0.5 μ m ), but no stimulation is seen with PCM-II d . In addition, earlier studies with PCM-I showed that, unlike either PCM-II or PCM-II d polylysine biphasically stimulated the dephosphorylation of both phosphorylase a and the myosin light chains. Nevertheless, in spite of these obvious differences between the enzymes other data suggests that the PCM-phosphatases may be related to each other. Incubation of PCM-II at 90°C for 10 min apparently releases heat-stable regulatory proteins which reverse the modulatory effects of polylysine on light chain and phosphorylase phosphatase activities expressed by either PCM-II or PCM-I. Similarly, heat-stable proteins released from PCM-I also reverse polylysine-mediated modulation of both PCM-I and PCM-II. These findings are consistent with a working hypothesis suggesting that PCM-phosphatase may consist of a modulatory domain containing several regulatory proteins and a catalytic domain. Interaction between the regulatory proteins and polycationic effectors could result in conformational changes such that phosphatase activity expressed against one substrate is enhanced, while activity expressed against a different substrate is reduced. Within this framework, the appearance of multiple forms of PCM-phosphatase, as well as the specific response of each form to polycationic modulation of relative substrate specificity, may be attributable to selective loss or acquisition of one or more regulatory proteins. Although further studies are required to test this hypothesis it provides a useful working model for interpreting some of our data and for projecting future experiments. Conceivably, modulation of PCM-phosphatase activity and relative substrate specificity by polycationic effectors and/or specific regulatory proteins may be an important mechanism for coordinating different cellular mechanisms such as arterial metabolism and contractility.

The effects of angiotensin I (0.2-3.2 µg) and angiotensin II (0.1-1.6 µg) injections into the pump-perfused arterial supply of the liver were studied in dogs anesthetized with sodium pentobarbital. Marked increases in hepatic artery perfusion pressure (10-50%), reflecting directionally similar changes in resistance to blood flow, were caused by either angiotensin I or angiotensin II. Resistance increases produced by angiotensin I were significantly attenuated by the synthetic nonapeptide SQ 20881 (Pyr-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro, 50 µg/kg, iv) that inhibits enzymatic conversion of angiotensin I to angiotensin II. In contrast, responses caused by angiotensin II were unaltered by SQ 20881. However, resistance increases caused by either angiotensin I or angiotensin II were blocked by 1-Sar-8-Ala-angiotensin II (100 µg/kg min -1 , ia), a specific angiotensin II antagonist. These findings parallel the finding that responses to angiotensin I in the vasculature supplied by the hepatic artery are largely caused by local enzymatic conversion of angiotensin I to angiotensin II. Such conversion appears to occur to the extent of about 46%.

Effects of close intra-arterial injections of angiotensins I and II on changes in renal blood flow and renal vascular resistance were studied in dogs anesthetized with pentobarbital sodium. In the first series of experiments, angiotensin I (0.375 µg) or angiotensin II (0.13 µg) decreased renal blood flow and pressures in a small renal cortical vein and the renal vein. Although renal vascular resistance was markedly increased by angiotensins I or II, these increases did not appear to involve renal venous segments. Responses to angiotensin I occurred when the injected agonist was not allowed to reach the systemic circulation, and were virtually abolished during infusion of SQ 20475, a synthetic pentapeptide (Pyr-Lys-Try-Ala-Pro) that inhibits enzymatic conversion of angiotensin I to angiotensin II. In the second series of experiments, renal vascular responses to intra-arterial injections of angiotensin I (0.375-3.75 µg) and angiotensin II (0.13-1.00 µg) were studied. Responses to angiotensin I, but not those to angiotensin II, were significantly attenuated by SQ 20475. The results suggest that similar renal vascular segments respond to angiotensins I and II. Responses to angiotensin I are probably due to its intrarenal conversion to angiotensin II. Such conversion appears to occur to the extent of about 4%.

The water-soluble proteinpolysaccharide of bovine nasal cartilage, called PPL, prevents sedimentation of freshly precipitating calcium phosphate below 1,000 x g but not above 10,000 x g. By a method recently described, PPL can be separated into several fractions of which the main ones are PPL-3 and PPL-5. PPL-5, but not PPL-3, resembles PPL in its capacity to inhibit calcium phosphate sedimentation. Precipitation of calcium phosphate in the presence of PPL can be used to separate it into three fractions: the first is associated with calcium phosphate sedimentable below 1,000 x g and resembles PPL-6; the second is associated with calcium phosphate sedimentable above 10,000 x g and resembles PPL-5; the third remains in the supernatant solution and is identical with PPL-3. Thus different fractions of PPL have widely different effects on calcium phosphate precipitation, and, conversely, calcium phosphate precipitation in the presence of PPL can separate it into different fractions. By the procedures described, an entity can be isolated containing 16% PPL-5 and 43% Ca3(PO4)2. Evidence is given which suggests that this may be a chemical compound. Adsorption of different forms of PPL to solid Ca3(PO4)2 seems to be nonspecific and not related to the effect described.

Actin-myosin interaction in aortic actomyosin reportedly requires phosphorylation of the 20,000 dalton myosin light chains. A spontaneously active phosphatase which dephosphorylates phosphorylase a and isolated phosphorylated cardiac myosin light chains was extracted from bovine aortic smooth muscle. This enzyme, when added to aortic native actomyosin (a) significantly suppressed phosphorylation of the light chains of the native hexameric smooth muscle myosin, (b) accelerated the rate and increased the magnitude of myosin light chain dephosphorylation in actomyosin that had been prephosphorylated, and (c) markedly attenuated the rate of actin-myosin interaction. These results support the hypothesis that myosin phosphorylation and subsequent actin-myosin interactions (contractility) in vascular smooth muscle may be modulated by spontaneously active aortic phosphatase.

Vascular smooth muscle contractility is tightly coupled to ATP production by intermediary metabolism. To elucidate mechanisms underlying coordination of metabolism and contractility we studied the time course of isometric force, and the activation of phosphorylase and cAMP-dependent protein kinases during stimulation of bovine coronary arterial strips with KCl. Isometric force reached a maximum after 10 min of exposure to 30 mM KCl (ED90) and was sustained throughout the subsequent 20-min period of contraction. In contrast, activation of phosphorylase was biphasic: enzymic activity reached a maximum (176 +/- 10% of control) after 3 min of contraction and then, though remaining above control, activity declined to a lower level (135 +/- 7% of control). However, no change occurred in the activity ratios for cAMP-dependent protein kinase assessed in either the presence (type II isozyme) or absence (type I isozyme) of 0.5 M NaCl. These data suggest that the activation of phosphorylase during K+-induced contraction is independent of the cAMP system. The biphasic activation of phosphorylase may reflect transient changes in the intracellular concentration of Ca2+ or the activation of a phosphatase(s) during the response.

The histone-H1 and polylysine stimulated “latent” phosphorylase phosphatase, characterized by a molecular weight of 260,000 in gel filtration and 130,000 in sucrose density gradient centrifugation has been identified as a major inhibitor-1 phosphatase in vascular smooth muscle. Its substrate: protein phosphatase inhibitor-1, was also shown to be present in the same tissue. Following treatment with β-mercaptoethanol the enzyme dissociates into a lower molecular weight (38,000) form with a higher specific activity.

The Ca2+-regulatory mechanism for contraction of smooth muscle involves phosphorylation of the 20,000-Da myosin light chains. In the fully relaxed muscle the intracellular concentration of Ca2+ is low ( 10-6M). Subsequently, formation of the Ca2+-calmodulin complex results in activation of myosin light-chain kinase and phosphorylation of the regulatory light chains. In this form, actin-myosin interaction is activated resulting in hydrolysis of ATP and cross-bridge cycling. The physiological correlate of these biochemical events is contraction of the smooth muscle cell (see (1–4) for reviews). Although recent studies suggest that cross bridges ma...