Effect of NPPB on chloride (CI) transport in distal colon of potassium (K+) adapted rats

Secondary hyperaldosteronism enhances the rate of K secretion in distal colon, at least in part, through the stimulation of Na⁺-K⁺-Cl^- cotransport across the basolateral membrane. To maintain a constant intracellular Cl^- activity an increase in Cl^- transport out of the cell must be assumed. We explored, under amiloride 10^-4 M and short circuited conditions, conductive pathways for Cl^- exit in the distal colon of K⁺-adapted rats by means of a putative Cl^- channel blocker, NPPB (5-nitro-2(3-phenyl-propylamino-benzoate. Results prior to NPPB showed an increase in JCl^ras after K⁺ loading from 5.84 + 0.66 to 8.33 ±0.86 and JCl^8m from 4.77 ±0.55 to 8.16±0.96/μEq h“¹ cm”² (P < 0.001), when compared with controls. Net fluxes were not different between groups. Luminal NPPB in K⁺ adaptation resulted in a decrease of JCl^sm, from 7.85 ±1.5 to 6.69 ± 1.5 μEq h^-1 cm^-2 (P < 0.05). There were no changes in both unidirectional Cl^- fluxes in controls under luminal NPPB and in potential difference (V) and short-circuit current (I^-.) under any condition. Finally, K⁺ adaptation resulted in an increase of luminal cyclic AMP (cAMP) concentration (0.09 ±0.02 to 0.20 ±0.03 pmol 100 μ^-1, P < 0.05), when compared with control rats. The data may suggest a transcellular recycling of Cl^- and an activated NPPB inhibitable serosal to mucosal Cl^- pathway on luminal membrane in the K⁺ adapted state, possibly mediated by an increase in cAMP production.     

Regulation of cell cyclic AMP in medullary thick ascending limb of Henle in a rat model of chronic renal failure

Chronic renal failure (CRF) is accompanied by adaptive changes in electrolyte reabsorption in the thick ascending limb of Henle of surviving nephrons. To study the cellular mechanism of this adaptation, we measured intracellular cAMP in micro-dissected medullary thick ascending limb (mTAL) segments in rats with CRF. mTAL exhibited in CRF an increase of basal cAMP from 25.6 ± 10.0 in controls to 65.8 ± 11.3 fmol mm^?1 tubule in CRF (P < 0.05). Vasopressin and calcitonin stimulated mTAL adenylate-cyclase in a dose-dependent manner in controls but failed to stimulate in CRF. Likewise, maximal stimulation with 10^?3 M 3-isobutyl-1-methylxanthine (IBMX) plus 10^?5 M forskolin increased cAMP in controls to 63.0 ± 16.0 but not in CRF, where maximal stimulated values remained at 63.1 ± 18.8 fmol mm^?1 tubule (P NS). Alpha₂-adrenoreceptor activation with clonidine at concentrations ranging from 10^?8 to 10^?6 M diminished cAMP production by 37% in CRF (P < 0.05), whereas no differences were found in controls. Thus, the basal intracellular cAMP is increased in rat mTAL in CRF. The finding that neither forskolin nor vasopressin were able to further augment intracellular cAMP would suggest that stimulatory pathways of the adenylate-cyclase system are activated in the basal state. However, mTAL cells in CRF seem to retain the response of normal epithelium to inhibitory pathways such as the one mediated by alpha₂-adrenoreceptors.