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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.

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.

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.

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.