When exposed to hypoxic conditions, coronary arteries dilate, which is an important protective response. Although vessel sensitivity to oxygen is well documented, the mechanisms are not known with certainty. To further characterize the mechanisms of oxygen sensing in the coronary artery, we tested the major classes of hypotheses by measuring the effects of hypoxia on energetics, [Ca(2+)](i), K(+) channel function, and pH(i). Hypoxia relaxes porcine coronary arteries stimulated with either KCl or U46619. The extent of relaxation is dependent on both the degree and kind of stimulation. [Ca(2+)](i) was measured in endothelium-denuded arteries using fura 2-AM and ratiometric fluorescent techniques. At lower stimulus levels, hypoxia decreased both force and [Ca(2+)](i). Inhibitor studies suggest that K(Ca) and K(ATP) channels are not involved in the hypoxic relaxation, whereas K(V) channels may play a minor role, if any. Despite the hypoxia-mediated decrease in force, [Ca(2+)](i) was unchanged or increased at high levels of stimulation. Despite a marked increase in lactate content, pH(i) (measured with the ratiometric fluorescent dye BCECF) was also little affected by hypoxia. Measurement of the phosphagen and metabolite profile of freeze-clamped arteries with analytical isotachophoresis indicated that hypoxia increased lactate content by 4-fold and decreased phosphocreatine to 60% of control. However, neither ATP nor P(i) was affected by hypoxia. Interestingly, additional stimulation under hypoxia increased force but not ATP utilization, as estimated from measurements of anaerobic lactate production. Thus, surprisingly, the economy of force maintenance is increased under hypoxia. In porcine coronary artery, both Ca(2+)-dependent and, importantly, Ca(2+)-independent mechanisms are involved in hypoxic vasodilatation. For the latter, mechanisms involving either ATP, [Ca(2+)](i), pH(i), or P(i) cannot be invoked. This novel oxygen sensing mechanism involves a decreased Ca(2+) sensitivity.

Force generated by smooth muscle cells is believed to result from the interaction of actin and myosin filaments and is regulated through phosphorylation of the myosin regulatory light chain (LC(20)). The role of other cytoskeleton filaments, such as microtubules and intermediate filaments, in determining the mechanical output of smooth muscle is unclear. In cultured fibroblasts, microtubule disruption results in large increases in force similar to contractions associated with LC(20) phosphorylation (15). One hypothesis, the "tensegrity" or "push-pull" model, attributes this increase in force to the disruption of microtubules functioning as rigid struts to resist force generated by actin-myosin interaction (9). In porcine coronary arteries, the disruption of microtubules by nocodazole (11 microM) also elicited moderate but significant increases in isometric force (10-40% of a KCl contracture), which could be blocked or reversed by taxol (a microtubule stabilizer). We tested whether this nocodazole-induced force was accompanied by changes in coronary artery stiffness or unloaded shortening velocity, parameters likely to be highly sensitive to microtubule resistance elements. Few changes were seen, ruling out push-pull mechanisms for the increase in force by nocodazole. In contrast, the intracellular calcium concentration, measured by fura 2 in the intact artery, was increased by nocodazole in parallel with force, and this was inhibited and/or reversed by taxol. Our results indicate that microtubules do not significantly contribute to vascular smooth muscle mechanical characteristics but, importantly, may play a role in modulation of Ca(2+) signal transduction.

The Na(+)-K(+)-ATPase (NKA) can affect intracellular Ca(2+) concentration regulation via coupling to the Na(+)-Ca(2+) exchanger and may be important in myogenic tone. We previously reported that in mice carrying a transgene for the NKA alpha(2)-isoform in smooth muscle (alpha(2sm+)), the alpha(2)-isoform protein as well as the alpha(1)-isoform (not contained in the transgene) increased to similar degrees (2-7-fold). Aortas from alpha(2sm+) mice relaxed faster from a KCl-induced contraction, hypothesized to be related to more rapid Ca(2+) clearance. To elucidate the mechanisms underlying this faster relaxation, we therefore measured the expression and distribution of proteins involved in Ca(2+) clearance. Na(+)-Ca(2+) exchanger, sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA), and plasma membrane Ca(2+)-ATPase (PMCA) proteins were all elevated up to approximately fivefold, whereas actin, myosin light chain, and calponin proteins were not changed in smooth muscle from alpha(2sm+) mice. Interestingly, the corresponding Ca(2+) clearance mRNA levels were unchanged. Immunocytochemical data indicate that the Ca(2+) clearance proteins are distributed similarly in wild-type and alpha(2sm+) aorta cells. In studies measuring relaxation half-times from a KCl-induced contraction in the presence of pharmacological inhibitors of SERCA and PMCA, we estimated that together these proteins were responsible for approximately 60-70% of relaxation in aorta. Moreover, the percent contribution of SERCA and PMCA to relaxation rates in alpha(2sm+) aorta was not significantly different from that in wild-type aorta. The coordinate expressions of NKA and Ca(2+) clearance proteins without change in the relative contributions of each individual protein to smooth muscle function suggest that NKA may be but one component of a larger functional Ca(2+) clearance system.

Hypoxia (achieved by bubbling with N2 instead of O2) reduces the force of a KCl (40 mm)‐induced contracture to ?10 % of the control value in guinea‐pig taenia caeci. The underlying mechanism of this relaxation in response to hypoxia was investigated by measuring the major cell signalling parameters, intracellular Ca2+ concentration ([Ca2+]i) and myosin regulatory light chain (LC20) phosphorylation (MLC‐Pi), as well as intracellular pH (pHi), a factor often suggested to mediate hypoxic relaxation of muscle. [Ca2+]i, measured using the ratiometric fluorescent dye fura‐2, increased when 40 mm KCl was added to physiological saline solution (PSS) (peak value assigned 100%), and the steady state after 15 min was 92.8 %. There were no detectable decreases in [Ca2+]i during hypoxia. MLC‐Pi, measured using isoelectric focusing‐polyacrylamide gel electrophoresis and identified using Western blotting, increased from 9 % of the total LC20 in Ca2+‐free PSS to a peak value of 51 % in 40 mM KCl‐PSS. The steady‐state value in hypoxia of 43 % was not significiantly different from that in control oxygenated conditions at the same point in time. pHi, measured using the ratiometric fluorescent dye 2′,7′‐bis(carboxyethyl)‐5(6)‐carboxyfluorescein (BCECF), under quiescent conditions (Ca2+‐free PSS) was 7.23 and increased to 7.36 with 40 mM KCl. After imposition of hypoxia pHi remained unchanged despite the known increase in both lactate content and production. As [Ca2+]i and MLC‐Pi, key factors in activation, were not decreased by hypoxia and changes in pHi were minor, hypoxic relaxation in guinea‐pig taenia caeci appears to be directly related to energy limitation rather than any oxygen‐sensing mechanism.