The buffer barrier hypothesis, [Ca2+]i homogeneity, and sarcoplasmic reticulum function in swine carotid artery

It is well accepted that contractile agonist stimulation of smooth muscle induces Ca2+ release from a non-mitochondrial intracellular store (reviewed in Rembold, 1996). This store has been identified as the sarcoplasmic reticulum. 1,4,5-Inositol trisphosphate (1,4,5-IP3) is proposed to be the primary second messenger responsible for Ca2+ release. The sarcoplasmic reticulum also contains a Ca2+ pump, termed the sarco(endo)plasmic reticulum ATPase (SERCA), which appears to be primarily responsible for Ca2+ uptake into the sarcoplasmic reticulum. These data suggest that smooth muscle contains the necessary proteins for agonist stimulated Ca2+ release. However, the relative importance of Ca2+ release in the resultant contraction has yet to be determined. Additionally, it is possible that the sarcoplasmic reticulum has role(s) in the regulation of intracellular [Ca2+] ([Ca2+]i) beyond agonist-induced Ca2+ release. Smooth muscle sarcoplasmic reticulum is a complex three-dimensional tubular structure in the cytosol. Electron microscopy reveals the existence of a portion of the sarcoplasmic reticulum closely apposed near the plasma membrane (Devine et al. 1972). This region of close apposition may have a special role in regulation of [Ca2+]i because a number of Ca2+ transport proteins, such as the Na+-Ca2+ exchanger, are preferentially localized to this region (Villa et al. 1993; Moore et al. 1993). It is possible that Ca2+ flux through this region may be involved in Ca2+ influx or efflux. For example, Ca2+ influx across the plasma membrane may be important to refill the sarcoplasmic reticulum after Ca2+ release. Ca2+ could also flow in a reverse direction. When contractile stimuli are removed, the sarcoplasmic reticulum may pump Ca2+ into its lumen, transport it to near the plasma membrane, and release it into the subsarcolemmal cytoplasm so it can be exported into the extracellular space. This specialized region of cytoplasm between the sarcoplasmic reticulum and the plasma membrane may have a different [Ca2+] than the remaining cytoplasm. In 1977, Van Breemen hypothesized that there are functional [Ca2+]i domains inside smooth muscle cells: (1) a contractile domain encompassing most of the cytoplasm (hereafter termed [Ca2+]c), and (2) a subplasmalemmal domain (hereafter termed [Ca2+]pm) (Van Breemen, 1977; Van Breemen et al. 1986). The latter domain includes the region where the sarcoplasmic reticulum is closely apposed to the plasma membrane. According to this hypothesis, the [Ca2+]pm may, at times, be higher than [Ca2+]i. He also hypothesized that higher [Ca2+]pm may have a role in refilling the sarcoplasmic reticulum with Ca2+. Subsequently other investigators, working in other cell types, have hypothesized the existence of subplasmalemmal Ca2+ domains (Nagai et al. 1989; Rembold, 1989; Takemura & Putney, 1989; Lederer et al. 1990; Augustine & Neher, 1992). Evidence for focal increases in [Ca2+]i was inferred by comparing fura-2-estimated [Ca2+]i with [Ca2+]pm as measured with calcium-activated K+ (KCa) channel activity (Stehno-Bittel & Sturek, 1992; Yoshikawa et al. 1996), synaptic vessel fusion (Smith & Augustine, 1988; Augustine & Neher, 1992; Brose et al. 1992), or Ca2+ dependent Cl− currents (Osipchuk et al. 1990) in isolated patch clamped cells. Unfortunately, measurement of [Ca2+]pm with fluorescent dyes has been difficult because (1) the region is very small and (2) the fluorescent dyes, per se, act as mobile Ca2+ buffers which attenuate and change the spatial characteristics of the Ca2+ domains (Neher & Augustine, 1992). We recently developed a method to estimate whether cellular [Ca2+] is homogeneous (e.g. [Ca2+]pm=[Ca2+]c) or inhomogeneous (i.e. [Ca2+]pm > [Ca2+]c) in the cell. We measure [Ca2+]i with both aequorin and fura-2 (Rembold et al. 1995). This dual measurement allows estimation of relative [Ca2+]i homogeneity based on the characteristics of these two Ca2+ indicators (Yue & Wier, 1998). Aequorin is a Ca2+ sensitive protein that is very sensitive to increases in [Ca2+] that occur in small regions of the cell (e.g. [Ca2+]pm). In contrast, fura-2 is predominantly a measure of mean [Ca2+] (i.e. [Ca2+]c). The ratio of the observed aequorin and fura-2 signals is an index of the relative degree of [Ca2+]i homogeneity in intact swine carotid arterial tissues (Rembold et al. 1995). Increases in this ratio suggest a more inhomogeneous [Ca2+] distribution. The goal of this study was to evaluate the physiological roles of [Ca2+]i homogeneity and sarcoplasmic reticulum function in the regulation of arterial smooth muscle [Ca2+]i and contraction. In this study we tested four hypotheses: (1) that increases in [Ca2+]pm were primarily responsible for refilling of the sarcoplasmic reticulum with Ca2+; (2) that the sarcoplasmic reticulum maintains a [Ca2+] gradient within unstimulated smooth muscle cells, specifically that basal levels of Ca2+ influx increase [Ca2+]pm more than [Ca2+]c in unstimulated smooth muscle; (3) that the sarcoplasmic reticulum is important in removal of Ca2+ from the centre of the cell; and (4) that Ca2+ release from the sarcoplasmic reticulum is crucial for the initial phase of agonist-induced contractions.