Maria Rizzo

Specific blockade of slowly activating IsK channels by chromanols — impact on the role of IsK channels in epithelia

Chromanols, which were recently shown to inhibit cAMP‐mediated Cl− secretion in colon crypts via a blockade of a cAMP‐activated K+ conductance, were analyzed for their effects on distinct cloned K+ channels expressed in Xenopus oocytes. The lead chromanol 293B specifically inhibited I sK channels with an IC50 of 7 μmol/l without affecting the delayed rectifier Kv1.1 or the inward rectifier Kir2.1. Moreover, several other chromanols displayed the same rank order of potency for I sK inhibition as demonstrated in colon crypts. Finally, we tested the effects of the previously described I sK blocker azimilide on cAMP mediated Cl− secretion in rat colon crypts. Similar to 293B azimilide inhibited the forskolin induced Cl− secretion. These data suggest that I sK protein induced K+ conductances are the targets for the chromanol 293B and its analogues, and azimilide.

Upregulation of apical sodium-chloride cotransporter and basolateral chloride channels is responsible for the maintenance of salt-sensitive hypertension

We investigated which of the NaCl transporters are involved in the maintenance of salt-sensitive hypertension. Milan hypertensive (MHS) rats were studied 3 mo after birth. In MHS, compared with normotensive strain (MNS), mRNA abundance, quantified by competitive PCR on isolated tubules, was unchanged, both for Na+/H+ isoform 3 (NHE3) and Na+-K+-2Cl- (NKCC2), but higher (119%, n = 5, P < 0.005) for Na+-Cl- (NCC) in distal convoluted tubules (DCT). These results were confirmed by Western blots, which revealed: 1) unchanged NHE3 in the cortex and NKCC2 in the outer medulla; 2) a significant increase (52%, n = 6, P < 0.001) of NCC in the cortex; 3) alpha- and beta-sodium channels [epithelial Na+ channel (ENaC)] unaffected in renal cortex and slightly reduced in the outer medulla, while gamma-ENaC remained unchanged. Pendrin protein expression was unaffected. The role of NCC was reinforced by immunocytochemical studies showing increased NCC on the apical membrane of DCT cells of MHS animals, and by clearance experiments demonstrating a larger sensitivity (P < 0.001) to bendroflumethiazide in MHS rats. Kidney-specific chloride channels (ClC-K) were studied by Western blot experiments on renal cortex and by patch-clamp studies on primary culture of DCT dissected from MNS and MHS animals. Electrophysiological characteristics of ClC-K channels were unchanged in MHS rats, but the number of active channels in a patch was 0.60 +/- 0.21 (n = 35) in MNS rats and 2.17 +/- 0.59 (n = 23) in MHS rats (P < 0.05). The data indicate that, in salt-sensitive hypertension, there is a strong upregulation, both of NCC and ClC-K along the DCT, which explains the persistence of hypertension.

Amiloride-inhibitable Na+ conductance in rat proximal tubule

Abstractu2002Previous single-channel recordings from the luminal membrane of the rabbit proximal tubule have revealed amiloride-inhibitable Na+ channels of a characteristic conductance range. The present study aimed to pursue this issue in rat proximal tubule. Control rats were compared to those put on a low-Na+ diet or pretreated by triamcinolone injections (s.c.). Stimulation of Na+ absorption by glucocorticoids was verified by examining the transepithelial voltage in Ussing chamber studies of the distal colon. The membrane voltage (Vm) of isolated, in-vitro-perfused proximal tubule segments was measured in patch-clamp and impalement studies. It was found that amiloride hyperpolarized Vm significantly by 2.1 ± 0.9 mV (n = 26) in tubules of control rats, by 3.9 ± 0.7 mV (n = 12) in rats put on a low-Na+ diet and by 3.7 ± 1.0 mV (n = 17) in rats treated with glucocorticoids. The effect of amiloride was concentration dependent with a half-maximal effect at < 1 μmol/l. RT-PCR techniques were used to search for the presence of the α-, β- and γ-subunits of the epithelial Na+ channel in isolated oximal tubule segments. The presence of the respective mRNAs was verified. These data indicate that: (1) amiloride-inhibitable Na+ channels are present in rat proximal tubules; (2) the Na+ conductance may be upregulated by Na+ deprivation but is still very limited when compared to total cell conductance; (3) therefore, the contribution of Na+-channel-mediated absorption to total proximal Na+ absorption is probably small.