Enhanced response to AVP in the interlobular artery from the spontaneously hypertensive rat

Abstract

Arginine vasopressin (AVP) induces exaggerated intracellular free calcium (Cai2+) responses in preglomerular smooth muscle cells from young spontaneously hypertensive rats (SHR) due to increased density of the AVP V1a receptor. The intention of the present paper was to examine the relative contribution of afferent arterioles (AA) and interlobular artery (ILA) in AVP- and norepinephrine-induced calcium signaling. The kidneys were perfused with agar solution in vivo, and thin cortical slices were enzyme digested to produce isolated agar-filled vascular fragments. Calcium responses were recorded in fura 2-loaded cells by Ca2+ imaging. Diameter changes were measured after AVP stimulation and mRNA for V1a was measured on isolated vessel fragments. SHR had a significantly higher baseline calcium ratio and lower resting diameter compared with normotensive Wistar-Kyoto rats (WKY). Stimulation with AVP (10(-7) M) in ILA fragments from SHR induced a ratio increase of 0.49 +/- 0.09, significantly higher than the ratio increase in AA from SHR (0.20 +/- 0.03, P < 0.01) and in ILA from WKY (0.24 +/- 0.03, P < 0.01). Stimulation with norepinephrine (10(-7) M) induced responses homogeneously distributed between the segments and strains. Nifedipine treatment or removal of external calcium (Cao2+) reduced the norepinephrine-induced peak response. Both norepinephrine- and AVP-induced sustained responses were abolished after Cao2+ removal in SHR and WKY (P < 0.01). Measurements of V1a receptor mRNA on isolated segments showed a threefold increase in ILA from SHR. The present findings indicate that the exaggerated Ca2+ and contractile response to AVP in SHR is mainly mediated through ILA vasoconstriction.

Angiotensin II-induced calcium signaling in the afferent arteriole from rats with two-kidney, one-clip hypertension

“…The method for the fura ratio measurements and the identification of vessels have been described before (11). In short, the fura 2 ratio was measured using an inverted Olympus IX-70 with a ϫ40 UAPO objective.…”

“…The arterioles are not sieved, centrifuged, or shaken during the current isolation technique, and the vessels are filled with an elastic agarose gel that prevents structural deformation during contraction. We have earlier found this preparation well suited for measurements of changes in intracellular calcium (11). To our knowledge, Ca 2ϩ signaling in vascular smooth muscle cells from the clipped and nonclipped kidney in 2K1C has not been studied.…”

A low‐cost, scalable technique to study distal coronary arteriole function

“…Before recording, temperature (from room temperature: 2-5°C min À1 ) and Ca 2+ concentration (3 min steps: 10 À5 , 10 À4 and 10 À3 M) were increased gradually. Comparing lumen diameters with microsphere, experiments previously performed at our laboratory indicates that the agarose method produces maximally dilated vessels (Hansen et al 2005). Venules were rarely seen in the suspension, but could be discriminated from their cousins by much thinner vessel wall, protruding nuclei (in mice) and a spiralling pattern of the SMCs (in contrast to the parallel pattern shown in Figure 1c upper left) when encountered.…”

“…The culprit was a trypsin pollutant in the Clostridium-derived collagenase extract, which cleaved the extracellular domain of the vasopressin receptor (V 1a ) before experiments took place. When the caveat was finally discovered, cleavage was prevented by adding a small amount of dispase inhibitor (Hansen et al 2005). Pollutants are not uncommon in enzyme extracts and should always be kept in mind if unexpected behaviour is encountered.…”

“…This is advantageous as it ensures that regulatory data match functional data. Quantitative PCR (Hansen et al 2005), proteomics and in situ hybridization have also been performed on agarose preparation vessels, but the amount of arterioles needed (10-50) usually requires that several investigators must cooperate to collect diameter data in addition to these measures.…”