Rapid secretagogue-induced activation of Na+H+ exchange in rat parotid acinar cells. Possible interrelationship between volume regulation and stimulus-secretion coupling.

We demonstrate a rapid activation of the Na+/H+ exchanger in intact rat parotid acini in response to muscarinic (carbachol; K1/2 = 0.4 microM) and alpha-adrenergic (epinephrine; K1/2 = 0.1 microM) stimulation. This rapid activation is apparently distinct from the relatively "slow" activation of the exchanger (t1/2 greater than or equal to 5 min) reported previously (Manganel, M., and Turner, R. J. (1989) J. Membr. Biol. 111, 191-198). This rapid activation is not produced by treatment of acini with active diacylglycerol analogues nor prevented by protein kinase inhibitors, arguing against the involvement of protein kinase C-dependent processes. Stimulation of the exchanger is, however, produced by concentrations of ionomycin which yield intracellular calcium levels in the physiologic (secretagogue-induced) range. In addition, chelation of intracellular calcium with 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid blocks the effect of carbachol, but calmodulin antagonists are without effect. The possibility that the rapid activation of the Na+/H+ exchanger may be associated with cell shrinkage arising from carbachol-induced calcium mobilization is explored. In support of this suggestion we present evidence that: (i) the Na+/H+ exchanger is stimulated by shrinkage of these cells, (ii) the carbachol dose dependence of Na+/H+ exchange activation correlates well with that of shrinkage (but not with that of intracellular calcium levels), and (iii) maneuvers which blunt carbachol- or calcium-induced shrinkage also blunt activation of the exchanger. We suggest that this osmoregulatory response may play an important role in maintaining ionic homeostasis during the acinar fluid secretory process.

Transport of urate and p-aminohippurate in rabbit renal brush-border membranes.

The mechanisms involved in urate and p-aminohippurate (PAH) transport in the rabbit renal brush-border membrane were investigated through study of membrane vesicles. Transport of [14C]urate and [3H]PAH was measured by a rapid filtration method. As previously reported by others, no OH(-)-PAH exchanger could be demonstrated by imposing an outwardly directed OH- gradient (pHin 7.4, pHout 6). In contrast, an OH(-)-lactate exchanger (or H(+)-lactate cotransport) was demonstrated. In the presence of valinomycin and an inwardly directed K+ gradient, both [14C]urate and [3H]PAH vesicle uptake were stimulated, demonstrating a potential-driven transport of these two anions. Probenecid, PAH, or cold urate decreased potential-driven urate uptake, suggesting that this transport was facilitated by a specific transport mechanism. The potential-driven urate transport described here may play a role in the second step of urate secretion in rabbits, because rate (or PAH) is transported across the brush-border membrane from the negative interior of the cell to the more positive omen.

Agonist-induced activation of Na+/H+ exchange in rat parotid acinar cells is dependent on calcium but not on protein kinase C.

We demonstrated previously that Na+/H+ exchange activity was enhanced in rat parotid acinar cells following muscarinic (carbachol) or alpha-adrenergic (epinephrine) stimulation and that this activation of the exchanger was preserved in plasma membrane vesicles prepared from these cells (Manganel, M., and Turner, R. J. (1989) J. Membr. Biol. 111, 191-198). The carbachol dose dependence of this effect as determined here in intact acini is in good agreement with the carbachol dose dependence of the fluid secretory response in this tissue (K0.5 approximately 3 microM). In addition, we show here that the stimulation of the exchanger by secretagogues cannot be mimicked by active diacylglycerol analogues, nor can it be prevented by the protein kinase inhibitors H7 and HA1004, arguing strongly against the involvement of protein kinase C in this effect. However, stimulation of the exchanger is observed in both intact acini treated with the calcium ionophore A23187 and in vesicles prepared from these acini. Moreover, carbachol, epinephrine, and A23187 are without effect when extracellular calcium is not present during acinar pretreatment. These results indicate that the stimulation of the Na+/H+ exchanger studied here is a consequence of agonist (or A23187)-induced increases in intracellular calcium levels due to calcium influx from the extracellular solution. The calmodulin inhibitors trifluoperazine and W7 (10(-4) M) prevented the stimulation of the exchanger induced by carbachol or epinephrine, but W7 could not block the stimulation produced by A23187 arguing against the involvement of calmodulin in this effect. Taken together with previous data from this and other laboratories, these results strongly indicate that the Na+/H+ exchanger and its regulation are intimately involved in the fluid secretory response of the rat parotid.

Agonist-induced activation of Na+/H+ exchange in rat parotid acinar cells.

The present studies were designed to test our previous suggestion that Na+/H+ exchange was activated by muscarinic stimulation of rat parotid acinar cells. Consistent with this hypothesis, we demonstrate here that intact rat parotid acini stimulated with the muscarinic agonist carbachol in HCO3- -free medium show an enhanced recovery from an acute acid load as compared to similarly challenged untreated preparations. Amiloride-sensitive 22Na uptake, due to Na+/H+ exchange, was also studied in plasma membrane vesicles prepared from rat parotid acini pretreated with carbachol. This uptake was stimulated two-fold relative to that observed in vesicles from control (untreated) acini. This stimulation was time dependent, requiring approximately 15 min of acinar incubation with carbachol to reach completion, and was blocked by the presence of the muscarinic antagonist atropine (2 x 10(-5) M) in the pretreatment medium. The effect of carbachol was dose dependent with K0.5 approximately 3 x 10(-6) M. Stimulation of the exchanger was also seen in vesicles prepared from acini pretreated with the alpha-adrenergic agonist epinephrine, but not with the beta-adrenergic agonist isoproterenol, or with substance P. Kinetic analysis indicated that the stimulation induced by carbachol was due to an alkaline shift in the pH responsiveness of the exchanger in addition to an increased apparent transport capacity. Taken together with previous results from this and other laboratories, these results strongly suggest that the Na+/H+ exchanger and its regulation are intimately involved in the fluid-secretory response of the rat parotid.

Coupled Na+/H+ exchange in rat parotid basolateral membrane vesicles.

pH gradient-dependent sodium transport in highly purified rat parotid basolateral membrane vesicles was studied under voltage-clamped conditions. In the presence of an outwardly directed H+ gradient (pHin = 6.0, pHout = 8.0) 22Na uptake was approximately ten times greater than uptake measured at pH equilibrium (pHin = pHout = 6.0). More than 90% of this sodium flux was inhibited by the potassium-sparing diuretic drug amiloride (K1 = 1.6 microM) while the transport inhibitors furosemide (1 mM), bumetanide (1 mM), SITS (0.5 mM) and DIDS (0.1 mM) were without effect. This transport activity copurified with the basolateral membrane marker K+-stimulated p-nitrophenyl phosphatase. In addition, 22Na uptake into the vesicles could be driven against a concentration gradient by an outwardly directed H+ gradient. pH gradient-dependent sodium flux exhibited a simple Michaelis-Menten-type dependence on sodium concentration consistent with the existence of a single transport system with KM = 8.0 mM at 23 degrees C. A component of pH gradient-dependent, amiloride-sensitive sodium flux was also observed in rabbit parotid basolateral membrane vesicles. These results provide strong evidence for the existence of a Na+/H+ antiport in rat and rabbit parotid acinar basolateral membranes and extend earlier less direct studies which suggested that such a transporter was present in salivary acinar cells and might play a significant role in salivary fluid secretion.

Transport of organic cations in brush border membrane vesicles from rabbit kidney cortex.

The transport of three organic cations, tetraethylammonium (TEA), morphine and N1-methylnicotinamide (NMN) was studied in brush border membrane vesicles from rabbit kidney cortex under voltage clamp conditions. A proton gradient (pHi = 6.0, pHo = 7.4) produced a large stimulation of TEA and morphine uptake, yielding a transient overshoot of 190 and 220% respectively, as compared to equilibrium uptake values. No overshoot was observed under pH equilibrium conditions (pHi = pHo = 7.4, control). These data suggest the presence of a proton-organic cation exchange mechanism in the rabbit renal cortical brush border membrane. Identical experimental conditions (proton gradient) failed however to stimulate significantly NMN transport above control values measured under pH equilibrium conditions. Proton gradient driven TEA transport showed an inhibition of 21% in the presence of NMN (1 mM) and of 63% in the presence of TEA (1 mM), and TEA transport was stimulated by preloading the vesicles with 1 mM TEA (305%) but not with 1 mM NMN (128%). NMN transport showed an inhibition of 39% in the presence of 1 mM TEA and of 27% in the presence of 1 mM NMN and its transport was stimulated by preloading the vesicles with 1 mM TEA (228%) and 1 mM NMN (178%). Our data suggest that TEA, NMN and morphine are transported by a common transport mechanism for which NMN has only a low affinity.

Sodium-pyrazinoate cotransport in rabbit renal brush border membrane vesicles.

Pyrazinoate (PZA) is an organic anion actively reabsorbed and secreted in the mammalian kidney. In experiments with rabbit renal brush border membrane vesicles, we characterized a sodium-PZA cotransport mechanism that could be involved in reabsorption. An inwardly directed sodium gradient stimulated the influx of PZA. The sodium-dependent transport was electroneutral, suggesting a 1:1 stoichiometry. The kinetic constants for sodium-PZA cotransport were measured under initial linear flux and zero trans conditions for both sodium and PZA. The apparent Km for sodium was about 60 mM. At 90 mM sodium the apparent Km for PZA was about 1.1 mM; increasing the sodium concentration augmented the apparent affinity for PZA. Cis inhibition of sodium-dependent PZA uptake was observed by the addition of nicotinate, lactate, probenecid, succinate, beta-hydroxybutyrate, and salicylate. Urate had no effect. [14C]PZA uptake was trans stimulated by PZA itself, lactate, and nicotinate. PZA shares a transport system(s) involved in the proximal tubular reabsorption of these two anions.

Blood pressure overshoot under pentobarbitone anaesthesia following a single dose of clonidine in rats.

1. Withdrawal of chronic antihypertensive therapy with clonidine is known to produce a blood pressure overshoot. It has been reported that the same may occur after a single dose of clonidine. 2. A single, intramuscular dose of clonidine (0.05 mg/kg) produced an overshoot in blood pressure, on the day following administration, in normotensive rats anaesthetized with sodium pentobarbitone. 3. No rebound elevation of mean arterial pressure or of heart rate occurred in conscious, normotensive, spontaneously hypertensive or renal hypertensive rats following this dose of clonidine, nor did it occur in rats anaesthetized with ether. 4. It is suggested that the overshoot phenomenon in rats under barbiturate anaesthesia may involve an interaction between an effect of clonidine and the barbiturate.