The Na+/Ca2+ exchanger, driven by a transmembrane Na+ gradient, plays a key role in regulating Ca2+ concentration in many cells. Although the exchanger influences Ca2+ concentration, its activity in smooth muscle appears to be closely coupled to Ca2+ availability from intracellular stores. This linkage might result if the exchanger were positioned close to Ca2+ storage sites within the sarcoplasmic reticulum. To test this hypothesis we have developed methods to assess the relative three-dimensional distribution of proteins involved in Na+/K+ pumping, Na+/Ca2+ exchange, Ca2+ storage within the sarcoplasmic reticulum, and attachment of contractile filaments to the membrane in smooth muscle. Here we report that the Na+/Ca2+ exchanger is largely co-distributed with the Na+/K+ pump on unique regions of the plasma membrane in register with, and close to, calsequestrin-containing regions of the sarcoplasmic reticulum in sites distinct from the sites where contractile filaments attach to the membrane. This molecular organization suggests that the plasma membrane is divided into at least two functional domains, and appear to provide a mechanism for the strong linkage seen in smooth muscle between Na+/K+ pumping and Na+/Ca2+ exchange, and between Na+/Ca2+ exchange and Ca2+ release from the sarcoplasmic reticulum.

We investigated whether myosin light chain phosphatase activity changes during nitric oxide-induced relaxation of contracted intact carotid media and how changes in phosphatase activity mediate this relaxation. We also investigated one mechanism for regulating this phosphatase. Myosin phosphatase activity, myosin light chain phosphorylation, guanosine 3,5-cyclic monophosphate (cGMP) concentration, and phosphorylation of the inhibitory protein CPI-17 were all assayed in homogenates of one carotid media ring at each time point during nitric oxide-induced relaxation. The application of sodium nitroprusside to histamine-contracted media caused rapid declines in light chain phosphorylation and force. These were temporally correlated with a rapid elevation of cGMP and a large transient increase in myosin phosphatase activity. During the early response to nitroprusside, when force declined, increases in myosin phosphatase activity, concurrent with cGMPmediated decreases in calcium and myosin light chain kinase activity, could accelerate light chain dephosphorylation. CPI-17 was dephosphorylated upon application of nitroprusside at the same time that myosin phosphatase activity increased, suggesting that the removal of inhibition by phospho-CPI-17 contributed to the increase in myosin phosphatase activity. After 20 min of nitroprusside, myosin phosphatase activity had declined to basal levels, however low force was sustained. Additional light chain phosphorylation-independent mechanisms may be involved in sustaining the relaxation.The phosphorylation of Ser-19 on the 20-kDa myosin regulatory light chains (MRLC) 1 is the primary determinant of cross-bridge attachment and cycling during contraction and relaxation in smooth muscle (1, 2). The extent of phosphorylation is determined by the balance of the activities of the Ca 2ϩ -calmodulin-activated myosin light chain kinase (MLCK) and the myosin light chain phosphatase (MLCP) (3). Initially MLCP was presumed to be constitutively active, and MRLC phosphorylation and steady-state force were reported to be proportional to myoplasmic [Ca 2ϩ ] (4). Relaxation occurred when a stimulus was removed, the myoplasmic [Ca 2ϩ ] and MLCK activity were reduced, and MRLC phosphorylation declined. However, relaxation can be induced in the presence of excitatory stimuli without proportional decreases in steadystate MRLC phosphorylation by various treatments (5-9) including nitrovasodilators. These relaxations could involve a change in MLCP activity.Nitric oxide released from endothelial cells, nonadrenergic noncholinergic inhibitory innervation, or exogenous nitrovasodilators is an important inhibitory agent in smooth muscle. Nitric oxide activates soluble guanylyl cyclase to generate cGMP (10). Elevated [cGMP] presumably activates cGMP-dependent protein kinase (PKG). The phosphorylation of several intracellular mechanisms by PKG results in a reduction in myoplasmic [Ca 2ϩ ] (11). Nitrovasodilators such as sodium nitroprusside (SNP) and nitroglycerin cause a rapid relaxation of acti...

Ca2+-sensitive processes at cell membranes involved in contraction, secretion, and neurotransmitter release are activated in situ or in vitro by Ca2+ concentrations ([Ca2+]) 10-100 times higher than [Ca2+] METHODSSmooth muscle cells were enzymatically isolated from stomachs of the toad Bufo marinus according to described procedures (26). Measuring Spectral Properties. Excitation spectra of membrane-associated FFP18 at various [Ca2+] were recorded using a SPEX spectrofluorimeter. Emission was collected at 510 nm.FFP18 was applied to the external surface of cells by 10-to 30-min incubations in amphibian physiological saline (APS) containing 0.5-10 ,uM FFP18. Cell suspensions with different free [Ca2+] levels were produced by a described procedure (25). The concentration of membrane-associated FFP18 and the density of FFP18 molecules on the cell membrane were estimated using described methods (25).Abbreviations: SR, sarcoplasmic reticulum; [Ca+2]j, intracellular free Ca2+ concentration; APS, amphibian physiological saline. 5368The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Although a role for the ryanodine receptor (RyR)

Serine 19 phosphorylation of the myosin regulatory light chain (MRLC) appears to be the primary determinant of smooth muscle force development. The relationship between MRLC phosphorylation and force is nonlinear, showing that phosphorylation is not a simple switch regulating the number of cycling cross bridges. We reexamined the MRLC phosphorylation-force relationship in slow, tonic swine carotid media; fast, phasic rabbit urinary bladder detrusor; and very fast, tonic rat anococcygeus. We found a sigmoidal dependence of force on MRLC phosphorylation in all three tissues with a threshold for force development of approximately 0.15 mol P(i)/mol MRLC. This behavior suggests that force is regulated in a highly cooperative manner. We then determined whether a model that employs both the latch-bridge hypothesis and cooperative activation could reproduce the relationship between Ser(19)-MRLC phosphorylation and force without the need for a second regulatory system. We based this model on skeletal muscle in which attached cross bridges cooperatively activate thin filaments to facilitate cross-bridge attachment. We found that such a model describes both the steady-state and time-course relationship between Ser(19)-MRLC phosphorylation and force. The model required both cooperative activation and latch-bridge formation to predict force. The best fit of the model occurred when binding of a cross bridge cooperatively activated seven myosin binding sites on the thin filament. This result suggests cooperative mechanisms analogous to skeletal muscle that will require testing.