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Outer medullary descending vasa recta (OMDVR) were dissected from the outer medullary vascular bundles of young rats, perfused in vitro, and loaded with fura 2 for measurement of intracellular calcium concentration ([Ca2+]i) by fluorescent ratio imaging. Fluorescent video images revealed that fura 2 selectively loads into endothelial cells but not pericytes. Bradykinin (BK), at concentrations > 10(-11) M, elicited an increase in [Ca2+]i from baseline values in the range from 50 to 100 nM to peak values of 600-800 nM followed by a sustained plateau of 150-250 nM. The vasopressin V1-receptor agonist [Phe2,Ile3,Orn8]vasopressin constricted OMDVR but yielded no observable [Ca2+]i response, a finding that is consistent with an endothelial cell origin for the fura 2 fluorescent signal. The BK [Ca2+]i response was blocked by the selective BK B2-receptor antagonists D-Arg-[Hyp3,Thi5.8,D-Phe7]BK and D-Arg-[Hyp3,D-Phe7,Leu8]BK but not the B1 antagonist des-Arg9-[Leu8]BK. BK vasodilated microperfused OMDVR that had been preconstricted with 10(-8) M angiotensin II. We conclude that the [Ca2+]i response of OMDVR endothelia can be selectively studied with fura 2, that BK increases endothelial [Ca2+]i via the B2 receptor, and that BK can vasodilate descending vasa recta.

The short-circuit current technique was used to examine the effects of N2-L-lysylbradykinin (LBK) on chloride secretion in the mucosae of the mouse intestine. It was found to be a potent chloride secretagogue in the mucosa lining the colon, jejunum, and cecum, as it is in most mammals, with 2 nM being sufficient to cause half-maximal secretion. The extent of the responses was in the order cecum > colon > jejunum. In cystic fibrosis (CF) null mice, with no CF transmembrane conductance regulator (CFTR) chloride channels, LBK caused no chloride secretion, but transporting activities for other ions were revealed. Introduction of the human CF gene into the genome of CF null mice at the zygote stage restored the chloride secretory activity of LBK, with only minor differences in potency. In mice in which the kinin B2 receptor gene had been disrupted, LBK had no effect, whereas the responses to forskolin were unchanged. Thus the acute effects of kinins on chloride secretion depend uniquely on kinin B2 receptors and CFTR chloride channels, which form the primary and final effector mechanisms of the secretory process.

Responses to T-kinin, a peptide formed from the acute-phase substrate T-kininogen, were investigated in the hindlimb vascular bed of the cat. Under constant-flow conditions, injections of T-kinin into the perfusion circuit in doses of 0.03-1 nmol induced rapid dose-related decreases in perfusion pressure. Responses to T-kinin were similar in time course and magnitude to responses to bradykinin and kallidin and were inhibited by the kinin B2-receptor antagonist, Hoe-140. Responses to T-kinin were attenuated by an inhibitor of nitric oxide synthase and by tetraethylammonium chloride and were enhanced in duration by the guanosine 3',5'-cyclic monophosphate (cGMP) phosphodiesterase inhibitor zaprinast. Responses to T-kinin were not altered by inhibitors of K+(ATP) channels, by the cyclooxygenase pathway, or by muscarinic or beta-adrenergic-receptor antagonists. These data suggest that vasodilator responses to T-kinin are mediated by kinin B2-receptor-stimulated release of nitric oxide from the endothelium and increased smooth muscle cGMP levels. These results indicate that activation of K+(ATP) channels and muscarinic or beta-adrenergic receptors and the release of vasodilator prostaglandins are not involved in mediating the response to T-kinin in the hindlimb circulation of the cat.

Responses to kallidin, des-Arg9-bradykinin (DABK), and des-Arg10-kallidin (DAK) were investigated in the hindlimb vascular bed of the cat under constant-flow conditions. Injections of kallidin, DABK, and DAK into the hindlimb perfusion circuit produced dose-dependent vasodilator responses in the hindlimb vascular bed. Vasodilator responses to kallidin and bradykinin (BK) were similar in magnitude and time course, and both peptides were approximately 100-fold more potent than DABK or DAK. Responses to kallidin were decreased by the kinin B2 antagonist, HOE 140, whereas responses to DABK and DAK were reduced by des-Arg9[Leu8]BK, a kinin B1-receptor antagonist. N omega-nitro-L-arginine methyl ester (L-NAME) reduced vasodilator responses to kallidin, DABK, and DAK, whereas meclofenamate, atropine, and U-37883A, a vascular selective ATP-sensitive K+ (K+ATP) channel-blocking agent, did not alter responses to the three peptides. These data suggest that both kinin B1 and B2 receptors are normally present in the hindlimb vascular bed. These data also suggest that kinin B1 and B2 receptor-mediated vasodilator responses are mediated by the release of nitric oxide and that the activation of K+ATP channels or muscarinic receptors, or the release of vasodilator prostaglandins play little if any role in mediating responses to kallidin, DABK, or DAK in the hindlimb vascular bed of the cat.

Confluent monolayers of pig renal papillary collecting tubule (RPCT) cells were formed on Millipore filters coated with collagen. They were clamped in Ussing-type chambers and used to measure short-circuit current (SCC). The monolayers had low potentials (0.1 mV) with the basolateral side positive. Small inward currents flowed under short-circuit conditions. Increases in SCC were obtained following addition of a number of agents. Receptors associated with SCC changes were disposed as follows: for kinins (e.g., lysyl-bradykinin) they were present on both sides of the tissue, while those for arginine vasopressin and norepinephrine were present on the basolateral side only. Epithelia responded to PGE2 added to the apical or basolateral face of the tissue; application to one side prevented the response from the contralateral side. The tissues also responded to forskolin, an activator of adenylate cyclase, with a sustained inward current that was sensitive to furosemide. Similar sustained inward currents were recorded following exposure to 8-bromoadenosine-3',5'-cyclic monophosphate (BrcAMP). Responses to kinins were attenuated by inhibition of fatty acid cyclooxygenase with either indomethacin or piroxicam or by replacing chloride with impermeant ions. If the SCC was first increased with forskolin, BrcAMP, or norepinephrine, the kinin effects on SCC were either abolished or reversed. It is concluded that kinin can cause chloride secretion in RPCT monolayers, possibly via a prostaglandin or a prostaglandin-adenylate cyclase mechanism. Secondary effects of kinin, exposed by first raising tissue cAMP levels, are not precluded.

Kinin-induced secretion in the intestine is accompanied by marked increases in mucosal adenosine 3', 5'-cyclic monophosphate (cAMP) and prostanoids that undoubtedly contribute to the overall secretory response. The cellular site at which these effects are initiated is unclear although a recent hypothesis has suggested the epithelial cell as the target for kinin action (6). We have investigated the effects of kallidin on the phospholipase-prostanoid-cAMP pathway in whole ileal mucosa and in epithelial cells isolated from the same tissue in the rat. Kallidin (1 microM) stimulated a marked rise (24 to 30-fold) in PG (prostaglandin) E2 release from the serosal surface of stripped ileal mucosa within 1-2 min, which correlated closely with the rise in mucosal short-circuit current. Mucosal cAMP levels were also increased two to threefold by kallidin. However, kinins were unable to elicit effects under the same conditions in suspensions of viable epithelial cells. PGE2 release was unaffected by kallidin or bradykinin at concentrations up to 100 microM, whereas cAMP levels could be stimulated by forskolin and PGE2 but not by kinin. Studies of intestinal phospholipase A2 (PLA2) activity also suggest a nonepithelial site for kinin action. This enzyme is responsible for liberating arachidonic acid from cellular phospholipids and has been shown to be activated by kinins in several tissues. In the intestine, PLA2 activity was found to be concentrated within the subepithelium with significantly lower levels in the epithelium itself. In addition, kallidin was unable to influence phospholipid labeling (an indirect measure of PLA2 activity) in cells incubated with [14C]arachidonic acid.(ABSTRACT TRUNCATED AT 250 WORDS)