MINERALOCORTICOID REGULATION OF ION CONDUCTIVE PATHWAYS OF THE CORTICAL COLLECTING DUCT

Mineralocorticoids (DOCA) are known to increase Na('+) absorption and K('+) secretion in the rabbit cortical collecting duct (CCD). However, the mechanism of regulation of the apical and basolateral cell membranes and tight junction ion conductive pathways (G('a), G('b), and G('tj), respectively) by mineralocorticoids are only partially understood. Using electrophysiological techniques and microelectrodes it was demonstrated that the apical cell membrane contained a dominant Ba('2+) sensitive K('+) conductive pathway, G(,K)('a), and an amiloride sensitive Na('+) conductive pathway, G(,Na)('a). The basolateral membrane contained a dominant Cl('-) conductive pathway, G(,Cl)('b), and a significant Ba('2+) sensitive K('+) conductive pathway, G(,K)('b). Upon elevating the mineralocorticoid levels of rabbits with intact adrenal glands it was found that V('te) was significantly increased after 1 day with a further increase after 13-16 days. These results indicated both primary and secondary effects of mineralocorticoid elevation. After 1 day of DOCA treatment, G(,Na)('a), I(,Na)('a) and I(,K)('a) increased by more than 2-fold and were maintained at high levels after 13-16 days of DOCA treatment. Secondary (chronic) effects of mineralocorticoids were evident after 4 days or more of DOCA treatment. These included a significant increase in G(,K)('a) from 4.0 to 10.2 mS.cm('-2) and a hyperpolarization of V('b) by -20 mV after 4 days of treatment. After 13-16 days of DOCA treatment V('b) remained hyperpolarized at -98.1 mV and G('tj) decreased from 5.6 to 4.2 mS.cm('-2). The hyperpolarization of V('b) was due to an increase in electrogenic Na('+) pump activity as the pump current, I(,act)('b), increased significantly from 35.7 to 195.2 (mu)A.cm('-2). Whereas net passive K('+) current across the basolateral membrane, I(,K)('b), was near zero in the control group of animals, i.e., K('+) near equilibrium, I(,K)('b) was approximately -40 (mu)A.cm('-2) in chronic DOCA treated animals. These results demonstrate that the initial effect of mineralocorticoid elevation is to increase G(,Na)('a). The ensuing depolarization of the apical membrane increases the driving force for K('+) exit into the lumen. Between 1 and 4 days of elevation, G(,K)('a) more than doubles in magnitude and at the same time the electrogenic activity of the Na('+) pump increases. This results in a hyperpolarization of V('b) which increases the driving force for K('+) uptake from the bath to the cell through a basolateral membrane conductive pathway. After 13-16 days G('tj) decreases thereby serving to maintain high electrochemical gradients across the epithelium. Therefore, the long term effects of mineralocorticoid elevation on the CCD appear to be adaptive mechanisms that serve to maintain high levels of K('+) secretion and Na('+) absorption. ^

ASSESSMENT OF SKELETAL MUSCLE BLOOD FLOW AND GLUCOSE METABOLISM WITH POSITRON EMITTING RADIONUCLIDES

In order to evaluate factors regulating substrate metabolism in vivo positron emitting radionuclides were used for the assessment of skeletal muscle blood flow and glucose utilization. The potassium analog, Rb-82 was used to measure skeletal muscle blood flow and the glucose analog, 18-F-2-deoxy-2-fluoro-D-glucose (FDG) was used to examine the kinetics of skeletal muscle transport and phosphorylation.^ New Zealand white rabbits' blood flow ranged from 1.0-70 ml/min/100g with the lowest flows occurring under baseline conditions and the highest flows were measured immediately after exercise. Elevated plasma glucose had no effect on increasing blood flow, whereas high physiologic to pharmacologic levels of insulin doubled flow as measured by the radiolabeled microspheres, but a proportionate increase was not detected by Rb-82. The data suggest that skeletal muscle blood flow can be measured using the positron emitting K+ analog Rb-82 under low flow and high flow conditions but not when insulin levels in the plasma are elevated. This may be due to the fact that insulin induces an increase in the Na+/K+-ATPase activity of the cell indirectly through a direct increase in the Na+/H+pump activity. This suggests that the increased cation pump activity counteracts the normal decrease in extraction seen at higher flows resulting in an underestimation of flow as measured by rubidium-82.^ Glucose uptake as measured by FDG employed a three compartment mathematical model describing the rates of transport, countertransport and phosphorylation of hexose. The absolute values for the metabolic rate of FDG were found to be an order of magnitude higher than those reported by other investigators. Changes noted in the rate constant for transport (k1) were found to disagree with the a priori information on the effects of insulin on skeletal muscle hexose transport. Glucose metabolism was however, found to increase above control levels with administration of insulin and electrical stimulation. The data indicate that valid measurements of skeletal muscle glucose transport and phosphorylation using the positron emitting glucose analog FDG requires further model application and biochemical validation. (Abstract shortened with permission of author.) ^

Three strategically placed hydrogen-bonding residues convert a proton pump into a sensory receptor.

In haloarchaea, light-driven ion transporters have been modified by evolution to produce sensory receptors that relay light signals to transducer proteins controlling motility behavior. The proton pump bacteriorhodopsin and the phototaxis receptor sensory rhodopsin II (SRII) differ by 74% of their residues, with nearly all conserved residues within the photoreactive retinal-binding pocket in the membrane-embedded center of the proteins. Here, we show that three residues in bacteriorhodopsin replaced by the corresponding residues in SRII enable bacteriorhodopsin to efficiently relay the retinal photoisomerization signal to the SRII integral membrane transducer (HtrII) and induce robust phototaxis responses. A single replacement (Ala-215-Thr), bridging the retinal and the membrane-embedded surface, confers weak phototaxis signaling activity, and the additional two (surface substitutions Pro-200-Thr and Val-210-Tyr), expected to align bacteriorhodopsin and HtrII in similar juxtaposition as SRII and HtrII, greatly enhance the signaling. In SRII, the three residues form a chain of hydrogen bonds from the retinal's photoisomerized C(13)=C(14) double bond to residues in the membrane-embedded alpha-helices of HtrII. The results suggest a chemical mechanism for signaling that entails initial storage of energy of photoisomerization in SRII's hydrogen bond between Tyr-174, which is in contact with the retinal, and Thr-204, which borders residues on the SRII surface in contact with HtrII, followed by transfer of this chemical energy to drive structural transitions in the transducer helices. The results demonstrate that evolution accomplished an elegant but simple conversion: The essential differences between transport and signaling proteins in the rhodopsin family are far less than previously imagined.

Regulation of sodium/glucose cotransporter expression in LLC-PK$\sb1$ cells

Expression of the Na$\sp+$/glucose cotransporter (SGLT1), a differentiated function of the pig kidney epithelial cell line LLC-PK$\sb1$ derived from proximal tubule, was further investigated. The differentiation inducer hexamethylene bisacetamide (HMBA) and IBMX, an inhibitor of cAMP phosphodiesterase, each stimulated a significant increase in Na$\sp+$/glucose cotransport activity, levels of the 75 kD cotransporter subunit and steady-state levels of the SGLT1 message. The action of HMBA is associated with involvement of polyamines and protein kinase C, and is synergistic with cAMP. We provide evidence that cAMP-elevating agents increase Na$\sp+$/glucose cotransporter expression, at least in part, via a post-transcriptional mechanism. Two molecular species of SGLT1 mRNA (3.9 kb and 2.2 kb) are transcribed from the same gene in LLC-PK$\sb1$ cells and differ only in the length of the 3$\sp\prime$ untranslated region (3$\sp\prime$ UTR). cAMP elevation differentially stabilized the 3.9 kb SGLT1 transcript from degradation but not the 22 kb species. UV-cross-linking and label transfer experiments indicated that cyclic AMP elevation was associated with formation of a 48 kD protein complex with a specific domain within the 3$\sp\prime$ UTR of SGLT1 mRNA. The binding was competitively inhibited by poly (U) and other U-rich RNA species such as c-fos ARE, and modulated by a protein kinase A-mediated phosphorylation/dephosphorylation mechanism. The binding site was mapped to a 120-nucleotide 3$\sp\prime$ UTR sequence which contains a uridine-rich region (URE). Our study provides the first demonstration that renal SGLT1 is post-transcriptionally regulated by a phosphorylation/dephosphorylation mechanism, and provides a deeper insight into gene regulation of this physiologically important cotransporter. ^