To explore the role of the atrial natriuretic factor (ANF) system in the pathophysiology of hypertension we examined the binding kinetics of synthetic ANF to cultured vascular smooth muscle cells (VSMCs) derived from the spontaneously hypertensive rat (SHR) and two normotensive controls-the Wistar Kyoto (WKY) and American Wistar (W). The number of maximal binding sites (Bmax) per cell (mean +/- SEM; X10(3] were: SHR = 278.0 +/- 33.0, WKY = 28.3 +/- 7.1 and W = 26.6 +/- 4.2. The differences between the SHR and normotensive strains were significant at p less than 0.001. The equilibrium dissociation constant (Kd; X 10(-9)M) was higher in SHR VSMCs (0.94 +/- 0.14) than in WKY (0.22 +/- 0.09; p less than 0.01) and W (0.39 +/- 0.14; p less than 0.02) cells. The plasma levels of the immunoreactive ANF were higher in SHR than the normotensive controls. We suggest that the relatively greater ANF receptor density in cultured VSMCs of the SHR represents a response to the in vitro environment which is relatively more deficient in ANF for VSMCs of the SHR as compared with the normotensive rats. Thus, the capacity of the SHR VSMC to regulate ANF receptor density appears to be independent of the blood pressure level.

Khalil, F.; Fine, B.; Kuriyama, S.; Nakamura, A.; Hatori, N.; Nakamura, M.; Aviv, A. Author Information

The binding of ouabain and K+ to the Na+ pump were analyzed in serially passed cultured vascular smooth muscle cells (VSMCs) originating from spontaneously hypertensive (SH), Wistar-Kyoto (WKY), and American Wistar (W) rats. Our techniques have utilized analyses of displacement of [3H]ouabain by both unlabeled ouabain and K+ from specific binding sites on the VSMCs. We have found that each of the VSMC preparations from the three rat strains appeared to demonstrate one population of specific ouabain receptors (Na+ pumps); the number of Na+ pump units (mean +/- SE, expressed as 10(5) units/cell; number of observations indicated in parentheses) of both the SH and WKY rats was significantly lower than the number of Na+ pump units of W rat VSMCs [SH: 3.00 +/- 0.02 (231), WKY: 2.87 +/- 0.05 (245), and W: 3.62 +/- 0.04 (225)]; the equilibrium dissociation constant values (microM) for ouabain in VSMCs of SH and WKY rats were similar but were significantly higher than that of VSMCs derived from W rats [SH: 4.69 +/- 0.09 (231), WKY: 4.57 +/- 0.12 (245), and W: 3.69 +/- 0.17 (225)]; and among the VSMCs originating from the three rat strains, the apparent equilibrium dissociation constant value for K+ (mM) was the lowest in those of the SH rat [1.04 +/- 0.003 (143), compared with VSMCs of the WKY rat [1.54 +/- 0.006 (135)] and W rat [1.19 +/- 0.003 (136)]. Our previous studies have demonstrated increased passive Na+ and K+ transport rate constants of SH rat VSMCs compared with either W or WKY rat cells. These findings suggest the possibility of higher permeabilities of the SH cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Na+-K+ passive transport and activity of the Na+ pump were examined in serially passed cultured vascular smooth muscle cells originating from spontaneously hypertensive (SH), Wistar-Kyoto (WKY), and Wistar (W) rats. Measurements included 22Na+ and 86Rb+ (K+ analogue) uptake and washout rate constants as well as intracellular Na+ and K+ levels. The aforementioned variables were studied in cells subjected to either 2 mM Ca2+ or Ca2+-deficient media. In 2 mM Ca2+ medium, SH rat cells demonstrated the highest exchange (uptake and washout) rate constants for Na+ and Rb+ (K+) among cells of the three rat strains. At this extracellular Ca2+ concentration, the Na+ pump activity of SH rat cells was higher than that of WKY rat cells and was not different from that of W rat cells. Incubation in Ca2+-deficient medium resulted in increased magnitudes of Rb+ washout and Na+ uptake rate constants in all cell preparations associated with elevated intracellular Na+ concentrations and augmented activity of the Na+ pump. Under this condition, cells derived from SH rats showed the highest Na+ uptake and Rb+ washout rate constants associated with the highest Na+ pump activity. The increase in intracellular Na+ level in Ca2+-deficient medium was the highest in SH rat cells. These findings show that innate membrane defects and the response of the Na+ pump to these abnormalities can be demonstrated in in vitro-grown vascular smooth muscle cells of the SH rat.

The rate of postreplication repair of the B16-F1 and the B16-F10 variant clones was compared to the parent B16CL4 mouse melanoma cells in an attempt to correlate the postreplication repair efficiency with the metastatic potential of these melanoma cells. The rate of postreplication repair of the B16-F10 subline was 47% higher than that of the parent B16CL4 mouse melanoma cells and 20% higher than that of the B16-F1 cells. This higher rate of postreplication repair in the B16-F10 cells correlates with its higher metastatic potential. It was also of interest to notice that the rate of postreplication repair of the B16-F1 and the B16-F10 cells are comparable to their rate of replicon joining in non-irradiated cells, in contrast to the parent B16CL4 cells whose rate of post-replication repair was significantly lower than its rate of replicon joining.

This study explores the relationship between extracellular calcium (Cao) and Na+-K+ regulation as it particularly pertains to the activity of the Na+ pump in cultured vascular smooth muscle cells (VSMCs) originating from Sprague-Dawley rats. As compared with cells incubated in media containing 0.5, 2.0, or 4.0 mM calcium, when the Na pump is active, VSMCs incubated in a Ca-deficient medium show a marked increase in intracellular sodium and no significant change in intracellular potassium. Associated with the rise in intracellular sodium there is an augmented activity of the Na pump. When the Na pump is inhibited, VSMCs incubated in either high-Ca medium (Cao = 4.0 mM) or Ca-deficient medium manifest a greater decline in intracellular potassium than cells incubated in media containing 0.5 or 2.0 mM calcium. Furthermore, when the Na pump is inhibited, VSMCs incubated in a Ca-deficient medium exhibit higher intracellular sodium levels in comparison with their counterparts incubated in media containing calcium. Flux experiments indicate that the aforementioned changes reflect increased membrane permeabilities to Na+ and K+. It is concluded that by regulating the permeability of the VSMC membrane, Cao plays an important role in the intracellular Na+-K+ homeostasis and that its effect on the Na pump is mediated via perturbations in the intracellular Na+ and K+ concentrations.

The number of Na+ pump units (Bmax) and the equilibrium dissociation constant (Kd) for ouabain as well as parameters of K+ binding to the Na+ pump were examined in in vitro-grown vascular smooth muscle cells ( VSMC ) derived from Sprague-Dawley rats. The technique to measure these variables utilizes analyses of [3H]ouabain displacement from its VSMC receptors by nonlabeled ouabain and K+. The mean values for Bmax and Kd in the cultured VSMCs were 1.95 X 10(5) receptor sites per single VSMC and 2.68 X 10(-6) M, respectively. The equilibrium dissociation constant for K+ (Ki) was 0.92 mM. K+ binding to the cultured VSMCs demonstrated positive cooperativity with a Hill coefficient (n) of 1.78.