Although animal cells swell in hypotonic medium, their volume is subsequently regulated by a net loss of KCl via Ca2(+)-dependent channels. A rise in intracellular free calcium ([Ca2+]i) thus appears to be an initial event in the adaptation of external tonicity, although details of this mechanism are not known. To investigate cell volume regulation, we measured [Ca2+]i (by use of fura-2) and cell diameters in single cells of cultured renal proximal convoluted tubule. We found that a rapid rise in [Ca2+]i occurred after cells were exposed to hypotonic solution (250 mosM) from 95.8 +/- 3.8 to 468.2 +/- 24 nM (n = 16). The rise in [Ca2+]i was not observed in cells exposed to Ca2(+)-free medium, and exposure to isotonic high-K or low-Na medium did not elicit a rise in [Ca2+]i, suggesting that this rise was a result of Ca2+ influx and not via voltage-dependent Ca2+ influx or decrease of Ca2+ efflux via Na(+)-Ca2+ pump. Pretreatment of cells with pertussis toxin dose dependently blocked the rise in [Ca2+]i. The hypotonic solution enhanced accumulations of inositol tris- and tetra-phosphate after a 1-min exposure. Studies that measured cell diameters suggest that recovery of cell volume may include the rise in [Ca2+]i. These data suggest that the regulatory volume decrease of proximal tubule cells involves a pertussis toxin-sensitive guanine nucleotide binding protein-operated Ca2+ influx.

Previous data suggested an active Cl-conductance in the renal proximal convoluted tubule, although single channel conductance and regulation were not found. We have investigated the presence and regulation of the C1-channel in proximal convoluted tubules by patch clamp analysis. The current-voltage relationship of whole cells with 130 mM NaCI in the pipette was nonlinear. The addition of 1-34 PTH (10-8 M), forskolin, or cAMP significantly increased whole cell Cl-conductance. We found a single Cl-channel in excised apical membranes possessing conductance of 33 picosiemens (pS) at positive and 22.5 pS at negative potential, which was blocked by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (10-i M) and was selective to Cl-(Cl/Na = 10). The channel was activated by prolonged membrane depolarization, by a catalytic subunit of protein kinase A (PKA), or by purified kinase C (PKC), but not by Ca2" (1 pM) inside the membrane. During cell-attached patch clamping, the channel was similarly activated by PTH, phorbol ester, or dibutyryl cAMP in a dose-dependent manner. To investigate second messenger contributions to the PTH-action, the PTH-evoked channels were modified further by the subsequent addition of several blockers of the second messengers. This suggested that PKA and PKC were involved in Cl-channel activation. We therefore conclude that renal proximal convoluted tubule cells possess an apical C1-channel activated by PTH via the PKA and PKC pathways. (J. Clin. Invest. 1991. 88:735-742.)

. Characterization of the portal signal in a nonsteady hyperglycemic state in conscious dogs. Am J Physiol Endocrinol Metab 284: E148-E155, 2003. First published September 11, 2002 10.1152/ajpendo.00079.2002To characterize the "portal signal" in a nonsteady hyperglycemic state, the kinetic relationship between net hepatic glucose balance (NHGB) and either hepatic glucose load (HGL) or plasma insulin level was determined during glucose infusion using a catheter technique in 36 conscious dogs. Glucose was infused intraportally (Po group) and peripherally (Pe group) at 39, 56, and 83 mol ⅐ kg Ϫ1 ⅐ min Ϫ1 over 2 h. There was a linear relationship between mean NHGB and either mean HGL or plasma insulin levels at each rate in either delivery (HGL: Po r ϭ 0.99, Pe r ϭ 0.95; insulin: Po r ϭ 99, Pe r ϭ 0.79). The threshold levels for net hepatic glucose uptake were 3.8 and 11.7 mmol/l for plasma glucose and 65 and 392 pmol/l for plasma insulin, respectively. The slope of the regression line against the abscissa was four times larger in portal than in peripheral delivery (HGL: Po 0.20 vs. Pe 0.05, P Ͻ 0.05; insulin: Po 0.19 vs. Pe 0.04, P Ͻ 0.05). These results suggest that the portal signal overrules the threshold of glucose for hepatic uptake by increasing hepatic extraction rate in a nonsteady hyperglycemic state.glucose delivery route; hepatic glucose load; insulin dependency; hepatic glucose uptake NUMEROUS STUDIES HAVE DOCUMENTED that hepatic glucose uptake is dependent on hepatic glucose load (HGL), plasma insulin level, and route of glucose delivery. However, in most studies, either hyperinsulinemic or hyperglycemic clamps were used in the presence of constant glucose, insulin, glucagon, and/or somatostatin concentrations (1, 2, 6, 14-16). It has not been demonstrated yet whether the portal signal functions similarly in a nonsteady hyperglycemic state as in the case during a clamp state. Moore et al. (13) infused glucose at a gradually increasing rate via the portal and peripheral delivery routes in dogs, with insulin and glucagon being free to change. However, arterial and portal plasma glucose profiles were largely not different with time in either delivery. In many clamp studies, peripheral delivery was combined with portal delivery to keep hepatic load constant. However, the portal signal appears not to be associated with the magnitude of arterial-portal glucose gradients at high glucose load (16). Recently, it has been reported that somatostatin blocks activation of the glucose sensor (5). Furthermore, several studies have demonstrated that pulsatile insulin concentrations have greater effects on glucose uptake than a constant concentration (3,7,11,17), and the initial acute rise in plasma insulin provokes a decrease in postprandial hyperglycemia (4, 10). Against this background, the present studies were undertaken to characterize the portal signal in a nonsteady hyperglycemic state. To this end, we determined the kinetic relationship between hepatic glucose disposal and HGL, plasma insulin levels, arterial...

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