A method for assessment of the dynamic response of the arterial baroreflex.

AIM: The baroreflex is a key mechanism in cardiovascular regulation, and alterations in baroreceptor function are seen in many diseases, including heart failure, obesity and hypertension. We propose a new method for analysing baroreceptor function from continuous blood pressure (BP) and heart rate (HR) in both health and disease. METHODS: Forty-eight-hour data series of BP and HR were collected with telemetry. Sprague Dawley rats on standard chow (n = 11) served as controls, while rats on a high-fat, high-fructose (HFHC) diet (n = 6) constituted the obese-hypertensive model. A third group of rats underwent autonomic blockade (n = 6). An autoregressive-moving-average with exogenous inputs (ARMAX) model was applied to the data and compared with the α-coefficient. RESULTS: Autonomic blockade caused a significant reduction in the strength of the baroreflex as estimated by ARMAX [ARMAX- baroreflex sensitivity (BRS)] -0.03 ± 0.01 vs. -0.19 ± 0.04 bpm heartbeat-1) . Both methods showed a ~50% reduction in BRS in the obese-hypertensive group compared with control (body weight 531 ± 27 vs. 458 ± 19 g, P < 0.05; mean arterial pressure 119 ± 3 vs. 102 ± 1 mmHg, P < 0.05; ARMAX-BRS -0.08 ± 0.01 vs. -0.15 ± 0.01 bpm heartbeat-1 , P < 0.05; α-coefficient BRS 0.51 ± 0.07 vs. 0.89 ± 0.07 ms mmHg-1 , P < 0.05). The ARMAX method additionally showed the open-loop gain of the baroreflex to be reduced by ~50% in the obese-hypertensive group (-2.3 ± 0.3 vs. -4.1 ± 0.3 bpm, P < 0.05), while the rate constant was similar between groups. CONCLUSION: The ARMAX model represents an efficient method for estimating several aspects of the baroreflex. The open-loop gain of the baroreflex was attenuated in obese-hypertensive rats compared with control, while the time response was similar. The algorithm can be applied to other species including humans.

Role of renal vascular potassium channels in physiology and pathophysiology.

The control of renal vascular tone is important for the regulation of salt and water balance, blood pressure and the protection against damaging elevated glomerular pressure. The K+ conductance is a major factor in the regulation of the membrane potential (Vm ) in vascular smooth muscle (VSMC) and endothelial cells (EC). The vascular tone is controlled by Vm via its effect on the opening probability of voltage-operated Ca2+ channels (VOCC) in VSMC. When K+ conductance increases Vm becomes more negative and vasodilation follows, while deactivation of K+ channels leads to depolarization and vasoconstriction. K+ channels in EC indirectly participate in the control of vascular tone by endothelium-derived vasodilation. Therefore, by regulating the tone of renal resistance vessels, K+ channels have a potential role in the control of fluid homoeostasis and blood pressure as well as in the protection of the renal parenchyma. The main classes of K+ channels (calcium activated (KCa ), inward rectifier (Kir ), voltage activated (Kv ) and ATP sensitive (KATP )) have been found in the renal vessels. In this review, we summarize results available in the literature and our own studies in the field. We compare the ambiguous in vitro and in vivo results. We discuss the role of single types of K+ channels and the integrated function of several classes. We also deal with the possible role of renal vascular K+ channels in the pathophysiology of hypertension, diabetes mellitus and sepsis.

Clinical Research Abstracts of the British Equine Veterinary Association Congress 2015.

REASONS FOR PERFORMING STUDY: Hypoglycin A (HG) appears to cause atypical myopathy (AM), but to our knowledge, detection of HG in affected and unaffected horses and concurrently in plants that they were exposed to has not previously been reported. OBJECTIVES: To investigate HG in samples from horses exposed to Acer pseudoplatanus (European sycamore maple) and in such plant material, at the time of clinical cases of AM in the herd. STUDY DESIGN: Cross-sectional study. METHODS: Blood was collected from 2 horses with AM and 22 clinically healthy co-grazing horses in 2 Swedish farms within one week of onset of signs (May 2014) and one month later, after horses were moved to other pastures. Ten healthy control horses from unaffected farms were sampled once. Samaras, seedlings, flowers and leaves from Acer pseudoplatanus and from Acer platanoides L (Norway maple) were collected from affected pastures. Hypoglycin A was analysed using chemical derivatisation with dansyl chloride (DNS) and ultra high performance liquid chromatography-tandem mass spectrometry. Hypoglycin A was detected as derivatised compound HG-DNS [M+H]+ with selected reaction monitoring. RESULTS: Hypoglycin A was detected in the horses affected with AM, and also in 20 out of 22 co-grazing horses. One month later, a surviving case horse and 9/20 co-grazing horses were still positive for HG. Controls from other farms were negative for HG. Hypoglycin A was detected in plant material from Acer pseudoplatanus, but not from Acer platanoides L. CONCLUSIONS: Horses grazing in pastures with HG-containing Acer pseudoplatanus were positive for HG in blood, and some showed severe signs of myopathy. Ethical animal research: Ethical consent for blood sampling was granted (C113/11) and horse owners gave their informed consent to inclusion of horses in the study. SOURCE OF FUNDING: National Veterinary Institute, Sweden. Competing interests: None declared.

K(V)7.4 channels participate in the control of rodent renal vascular resting tone.

AIM: We tested the hypothesis that K(V)7 channels contribute to basal renal vascular tone and that they participate in agonist-induced renal vasoconstriction or vasodilation. METHODS: KV 7 channel subtypes in renal arterioles were characterized by immunofluorescence. Renal blood flow (RBF) was measured using an ultrasonic flow probe. The isometric tension of rat interlobar arteries was examined in a wire myograph. Mice afferent arteriolar diameter was assessed utilizing the perfused juxtamedullary nephron technique. RESULTS: Immunofluorescence revealed that K(V)7.4 channels were expressed in rat afferent arterioles. The K(V)7 blocker XE991 dose-dependently increased the isometric tension of rat interlobar arteries and caused a small (approx. 4.5%) RBF reduction in vivo. Nifedipine abolished these effects. Likewise, XE991 reduced mouse afferent arteriolar diameter by approx. 5%. The K(V)7.2-5 stimulator flupirtine dose-dependently relaxed isolated rat interlobar arteries and increased (approx. 5%) RBF in vivo. The RBF responses to NE or Ang II administration were not affected by pre-treatment with XE991 or flupirtine. XE991 pre-treatment caused a minor augmentation of the acetylcholine-induced increase in RBF, while flupirtine pre-treatment did not affect this response. CONCLUSION: It is concluded that K(V)7 channels, via nifedipine sensitive channels, have a role in the regulation of basal renal vascular tone. There is no indication that K(V)7 channels have an effect on agonist-induced renal vasoconstriction while there is a small effect on acetylcholine-induced vasodilation.

Mechanisms of K(+) induced renal vasodilation in normo- and hypertensive rats in vivo.

AIM: We investigated the mechanisms behind K(+) -induced renal vasodilation in vivo in normotensive Sprague-Dawley (SD) rats and spontaneously hypertensive rats (SHR). METHODS: Renal blood flow (RBF) was measured utilizing an ultrasonic Doppler flow probe. Renal vascular resistance (RVR) was calculated as the ratio of mean arterial pressure (MAP) and RBF (RVR = MAP/RBF). Test drugs were introduced directly into the renal artery. Inward rectifier K(+) (K(ir) ) channels and Na(+) ,K(+) -ATPase were blocked by Ba(2+) and ouabain (estimated plasma concentrations ∼20 and ∼7 µm) respectively. RESULTS: Confocal immunofluorescence microscopy demonstrated K(ir) 2.1 channels in pre-glomerular vessels of SD and SHR. Ba(2+) caused a transient (6-13%) increase in baseline RVR in both SD and SHR. Ouabain had a similar effect. Elevated renal plasma [K(+) ] (∼12 mm) caused a small and sustained decrease (5-13%) in RVR in both strains. This decrease was significantly larger in SHR than in SD. The K(+) -induced vasodilation was attenuated by Ba(2+) in control SD and SHR and by ouabain in SD. Nitric oxide (NO) blockade using l-NAME treatment increased MAP and decreased RBF in both rat strains, but did not affect the K(+) -induced renal vasodilation. CONCLUSION: K(+) -induced renal vasodilation is larger in SHR, mediated by K(ir) channels in SD and SHR, and in addition, by Na(+) ,K(+) -ATPase in SD. In addition, NO is not essential for K(+) -induced renal vasodilation.

Na+-independent, nifedipine-resistant rat afferent arteriolar Ca2+ responses to noradrenaline: possible role of TRPC channels.

AIM: In rat afferent arterioles we investigated the role of Na(+) entry in noradrenaline (NA)-induced depolarization and voltage-dependent Ca(2+) entry together with the importance of the transient receptor potential channel (TRPC) subfamily for non-voltage-dependent Ca(2+) entry. METHODS: R (340/380) Fura-2 fluorescence was used as an index for intracellular free Ca(2+) concentration ([Ca(2+)](i)). Immunofluorescence detected the expression of TRPC channels. RESULTS: TRPC 1, 3 and 6 were expressed in afferent arteriolar vascular smooth muscle cells. Under extracellular Na(+)-free (0 Na) conditions, the plateau response to NA was 115% of the baseline R(340/380) (control response 123%). However, as the R(340/380) baseline increased (7%) after 0 Na the plateau reached the same level as during control conditions. Similar responses were obtained after blockade of the Na(+)/Ca(2+) exchanger. The L-type blocker nifedipine reduced the plateau response to NA both under control (from 134% to 116% of baseline) and 0 Na conditions (from 112% to 103% of baseline). In the presence of nifedipine, the putative TRPC channel blockers SKF 96365 (30 µm) and Gd(3+) (100 µm) further reduced the plateau Ca(2+) responses to NA (from 117% to 102% and from 117% to 110% respectively). CONCLUSION: We found that Na(+) is not crucial for the NA-induced depolarization that mediates Ca(2+) entry via L-type channels. In addition, the results are consistent with the idea that TRPC1/3/6 Ca(2+) -permeable cation channels expressed in afferent arteriolar smooth muscle cells mediate Ca(2+) entry during NA stimulation.

Calcium handling in afferent arterioles.

The cytosolic intracellular calcium concentration ([Ca(2+)](i)) is a major determining factor in the vascular smooth muscle tone. In the afferent arteriole it has been shown that agonists utilizing G-protein coupled receptors recruit Ca(2+) via release from intracellular stores and entry via pathways in the plasma membrane. The relative importances of entry vs. mobilization seem to differ between different agonists, species and preparations. The entry pathway might include different types of voltage sensitive Ca(2+) channels located in the plasmalemma such as dihydropyridine sensitive L-type channels, T-type channels and P/Q channels. A role for non-voltage sensitive entry pathways has also been suggested. The importance of voltage sensitive Ca(2+) channels in the control of the tone of the afferent arteriole (and thus in the control of renal function and whole body control of extracellular fluid volume and blood pressure) sheds light on the control of the membrane potential of afferent arteriolar smooth muscle cells. Thus, K(+) and Cl(-) channels are of importance in their role as major determinants of membrane potential. Some studies suggest a role for calcium-activated chloride (Cl(Ca)) channels in the renal vasoconstriction elicited by agonists. Other investigators have found evidence for several types of K(+) channels in the regulation of the afferent arteriolar tone. The available literature in this field regarding afferent arterioles is, however, relatively sparse and not conclusive. This review is an attempt to summarize the results obtained by others and ourselves in the field of agonist induced afferent arteriolar Ca(2+) recruitment, with special emphasis on the control of voltage sensitive Ca(2+) entry. Outline of the Manuscript: This manuscript is structured as follows: it begins with an introduction where the general role for [Ca(2+)](i) as a key factor in the regulation of the tone of vascular smooth muscles (VSMC) is detailed. In this section there is an emphasis is on observations that could be attributed to afferent arteriolar function. We then investigate the literature and describe our results regarding the relative roles for Ca(2+) entry and intracellular release in afferent arterioles in response to vasoactive agents, with the focus on noradrenalin (NA) and angiotensin II (Ang II). Finally, we examine the role of ion channels (i.e. K(+) and Cl(-) channels) for the membrane potential, and thus activation of voltage sensitive Ca(2+) channels.

Reduced rat renal vascular response to angiotensin II after chronic inhibition of nNOS.

The neuronal isoform of nitric oxide synthase (nNOS) in the kidney is predominantly located in the macula densa (MD) cells. These cells are known to be the sensor in the tubuloglomerular feedback, which influences the tonus of the afferent arteriole. This study investigated the effect of angiotensin II (Ang II) after chronic inhibition of nNOS on renal blood flow (RBF) and cytosolic calcium concentration [Ca(2+)]i in smooth muscle cells from afferent arterioles. Measurements of RBF were made in two control groups and two groups treated with a nNOS inhibitor, 7-nitro indazole (7-NI), for 1 and 4 weeks. At the time of the experiment Ang II bolus was given in the renal artery before and during i.v. l-NNA. [Ca(2+)]i was measured in arterioles from control rats and from rats treated for 1 week with 7-NI. RBF decreased after bolus Ang II by 60 +/- 11% in the control vs. 23 +/- 8% in the 1 week 7-NI treated group. The decreased sensitivity to Ang II after 1 week of 7-NI treatment compared with control rats persisted after l-NNA infusion. There were no differences from control in the group treated for 4 weeks. Ang II gave a transient [Ca(2+)]i increase in vessels from control rats whereas this response was absent in 1 week 7-NI-treated rats. A possible explanation for these findings could be a down regulation of Ang II receptors. The renal vasculature of rats exhibits a diminished RBF and [Ca(2+)]i response to Ang II after 1 week blockade of nNOS.

Norepinephrine-induced calcium signaling pathways in afferent arterioles of genetically hypertensive rats.

This study provides new information about the relative importance of calcium mobilization and entry in the renal vascular response to adrenoceptor activation in afferent arterioles isolated from 7- to 8-wk-old Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). Intracellular free calcium concentration ([Ca(2+)](i)) was measured in microdissected arterioles utilizing ratiometric photometry of fura 2 fluorescence. There was no significant strain difference in baseline [Ca(2+)](i). Norepinephrine (NE; 10(-6) and 10(-7) M) elicited immediate, sustained increases in [Ca(2+)](i). The general temporal pattern of response to 10(-6) M NE consisted of an initial peak and a maintained plateau phase. The response to NE was partially blocked by nifedipine (10(-6) M) or 8-(N,N-diethylamino) octyl-3,4,5-trimetoxybenzoate (TMB-8; 10(-5) M). A calcium-free external solution abolished the sustained [Ca(2+)](i) plateau response to NE, with less influence on the peak response. In the absence of calcium entry, TMB-8 (10(-5) M) completely blocked the calcium response to NE in WKY but not SHR, suggesting strain differences in mobilization. A higher concentration of TMB-8 (10(-4) M), however, blocked all discernible mobilization in both strains. We conclude that there are differences in Ca(2+) handling in renal resistance vessels between young WKY and SHR with respect to mobilization stimulated by alpha-adrenoceptors. Afferent arterioles of young SHR appear to have a larger inositol-1,4,5-trisphosphate-sensitive pool or release from a site less accessible to TMB-8.

Alpha1-adrenoceptor subtypes on rat afferent arterioles assessed by radioligand binding and RT-PCR.

We utilized [3H]prazosin saturation and competition radioligand binding studies to characterize the expression of alpha1-adrenoceptors in preglomerular vessels. mRNA for adrenoceptor subtypes was assayed using RT-PCR. The vessels were isolated using an iron oxide-sieving method. [3H]prazosin bound to a single class of binding sites (Kd 0.087 +/- 0.012 nM, Bmax 326 +/- 56 fmol/mg protein). Phentolamine displaced [3H]prazosin (0.2 nM) with a pK(i) of 8.37 +/- 0.09. Competition with the selective alpha1A-adrenoceptor antagonist 5-methylurapidil fit a two-site model (pK(i) 9.38 +/- 0.21 and 7.04 +/- 0.15); 59 +/- 3% of the sites were high-affinity, and 41 +/- 3% were low-affinity binding sites. Competition with the alpha1D-adrenoceptor antagonist 8-(2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl)-8-azaspiro[4.5]decane-7,9-dione dihydrochloride (BMY-7378) fit a one-site model with low affinity (pK(i) 6.83 +/- 0.03). The relative contents of alpha1A-, alpha1B-, and alpha1D-adrenoceptor mRNAs were 64 +/- 5, 25 +/- 5, and 11 +/- 1%, respectively. Thus there was a very good correlation between mRNA and receptor binding for the subtypes. These data indicate a predominance of the alpha1A-adrenoceptor subtype in rat renal resistance vessels, with smaller densities of alpha1B- and alpha1D-adrenoceptors.

Conducted vasoconstriction in rat mesenteric arterioles: role for dihydropyridine-insensitive Ca(2+) channels.

The aim of this study was to evaluate the role of voltage-operated Ca(2+) channels in the initiation and conduction of vasoconstrictor responses to local micropipette electrical stimulation of rat mesenteric arterioles (28 +/- 1 microm, n = 79) in vivo. Local and conducted (600 microm upstream from the pipette) vasoconstriction was not blocked by TTX (1 micromol/l, n = 5), nifedipine, or nimodipine (10 micromol/l, n = 9). Increasing the K(+) concentration of the superfusate to 75 mmol/l did not evoke vasoconstriction, but this depolarizing stimulus reversibly abolished vasoconstrictor responses to current stimulation (n = 7). Addition of the T-type Ca(2+) antagonist mibefradil (10 micromol/l, n = 6) to the superfusate reversibly blocked local and conducted vasoconstriction to current stimulation. With the use of RT-PCR techniques, it was demonstrated that rat mesenteric arterioles <40 microm do not express mRNA for L-type Ca(2+) channels (alpha(1C)-subunit), whereas mRNA coding for T-type subunits was found (alpha(1G)- and alpha(1H)-subunits). The data indicate that L-type Ca(2+) channels are absent from rat mesenteric arterioles (<40 microm). Rather, the vasoconstrictor responses appear to rely on other types of voltage-gated, dihydropyridine-insensitive Ca(2+) channels, possibly of the T-type.

alpha(1)-adrenoceptor subtypes in rat renal resistance vessels: in vivo and in vitro studies.

This study provides new information about the relative importance of different alpha(1)-adrenoceptors during norepinephrine (NE) activation in rat renal resistance vessels. In Sprague-Dawley rats, we measured renal blood flow (RBF) using electromagnetic flowmetry in vivo and the intracellular free calcium concentration ([Ca(2+)](i)) utilizing ratiometric photometry of fura 2 fluorescence in isolated afferent arterioles. Renal arterial bolus injection of NE produced a transient 46% decrease in RBF. In microdissected afferent arterioles, NE (1 microM) elicited an immediate square-shaped increase in [Ca(2+)](i), from 90 to 175 nM (P < 0.001). Chloroethylclonidine (CEC) (50 microM) had no chronic irreversible alkylating effect in vitro but exerted acute reversible blockade on norepinephrine (NE) responses both on [Ca(2+)](i) in vitro and on RBF in vivo. The RBF response was attenuated by approximately 50% by the putative alpha(1A)-adrenoceptor and alpha(1D)-adrenoceptor antagonists 5-methylurapidil (5-MU), and 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4. 5]decane-7,9-dione dihydrochloride (BMY-7378) (12.5 and 62.5 microg/h), respectively. The in vitro [Ca(2+)](i) response to NE was blocked approximately 25% and 50% by 5-MU (100 nM and 1 microM). BMY-7378 (100 nM and 1 microM) attenuated the NE-induced response by approximately 40% and 100%. The degree of inhibition in vitro was similar to the in vivo experiments. In conclusion, 5-MU and BMY-7378 attenuated the NE-induced responses, although relatively high concentrations were required, suggesting involvement of both the alpha(1A)-adrenoceptor and alpha(1D)-adrenoceptor. Participation of the alpha(1B)-adrenoceptor is less likely, as we found no evidence for CEC-induced alkylation.

The influence of beta-adrenergic activation on noradrenergic alpha1 activation of rabbit afferent arterioles.

The aim of the present investigation was to examine the effect of beta-adrenergic stimulation on smooth muscle calcium concentration ([Ca2+]i) in resting conditions and after administration of norepinephrine (NE) to stimulate alpha-adrenoceptors in isolated rabbit afferent arterioles loaded with the calcium-sensitive fluorescent probe fura-2. [Ca2+]i was evaluated in the proximal and distal parts of the arterioles. NE (4x10(-7) M) increased [Ca2+]i in both these regions. The alpha1-adrenoceptor antagonist prazosin (10(-7) M) totally abolished the NE-induced increase in [Ca2+]i, while the alpha2-adrenoceptor antagonist yohimbine (5x10(-7) M) had no influence on the response to NE. When beta-adrenoceptors were stimulated, using isoproterenol (10(-7) M), the NE-induced increase in [Ca2+]i was significantly lower in both regions. Activation of beta-adrenoceptors with isoproterenol did not affect the [Ca2+]i increase in response to depolarization with K+. Since beta-adrenoceptor stimulation raises the smooth muscle cell levels of cAMP, an adenylate cyclase stimulator, forskolin (10(-5) M) was administered prior to NE application. This maneuver also blunted the increase in [Ca2+]i in both regions. We conclude that the calcium response to NE in the isolated rabbit afferent arteriole is mediated by an alpha1-adrenoceptor. beta-Adrenoceptor stimulation and forskolin blunt the increase in [Ca2+]i induced by NE stimulation, indicating that cAMP counteracts the NE-induced activation of alpha1-adrenoceptors.

Calcium recruitment in renal vasculature: NE effects on blood flow and cytosolic calcium concentration.

This study provides new information about the relative importance of Ca2+ mobilization and entry in the renal vascular response to adrenoceptor activation. We measured renal blood flow (RBF) in Sprague-Dawley rats in vivo using electromagnetic flowmetry. We measured intracellular free Ca2+ concentration ([Ca2+]i) in isolated afferent arterioles utilizing ratiometric photometry of fura-2 fluorescence. Renal arterial injection of NE produced a transient decrease in RBF. The response was attenuated, in a dose-dependent manner, up to approximately 50% by nifedipine, an antagonist of L-type Ca2+ entry channels. Inhibition of Ca2+ mobilization by 3,4, 5-trimethoxybenzoic acid-8-(diethylamino)octyl ester (TMB-8) inhibited the renal vascular effects of NE in a dose-dependent manner, with maximal blockade of approximately 80%. No additional attenuation was observed when nifedipine and TMB-8 were administered together. In microdissected afferent arterioles, norepinephrine (NE; 10(-6) M) elicited an immediate square-shaped increase in [Ca2+]i, from 110 to 240 nM. This in vitro response was blocked by nifedipine (10(-6) M) and TMB-8 (10(-5) M) to a degree similar to that of the in vivo experiments. A nominally calcium-free solution blocked 80-90% of the [Ca2+]i response to NE. The increased [Ca2+]i elicited by depolarization with medium containing 50 mM KCl was totally blocked by nifedipine. In contrast, TMB-8 had no effect. Our results indicate that both Ca2+ entry and mobilization play important roles in the renal vascular Ca2+ and contractile response to adrenoceptor activation. The entry and mobilization mechanisms activated by NE may interact. That a calcium-free solution caused a larger inhibition of the NE effects on afferent arterioles than nifedipine suggests more than one Ca2+ entry pathway.

Hypertension and impaired renal angiotensin II response in rats after chronic neuronal nitric oxide synthase inhibition.

Nitric oxide (NO) produced by the macula densa cells is important for the control of tubuloglomerular feedback (TGF). Reduced production of NO by these cells activates TGF and could result in hypertension, although the TGF activity is then normalized in the hypertensive state. The normalization of TGF in this form of hypertension might be explained by an impaired ability of angiotensin II to constrict renal vessels or by up-regulation of some other vasodilator not affected by NO synthase inhibitors.

Effects of stimulation and inhibition of protein kinase C on the cytosolic calcium concentration in rabbit afferent arterioles.

The effect of the protein kinase C (PKC) inhibitor chelerytrine (Ch) and the PKC activator 12-0-tetradecanoyl-phorbol-13-acetate (TPA) on the cytosolic calcium concentration ([Ca2+]i) in isolated intact rabbit afferent arterioles was investigated. [Ca2+]i was measured in the proximal and distal parts of the arteriole. Administration of 1 microM Ch gave rise to a peak followed by an elevated level of [Ca2+]i in both these parts. Neither the peak nor the elevated level of [Ca2+]i was significantly reduced by 1 microM nifedipine. The relative peak increase in [Ca2+]i in response to 1 microM noradrenaline (NA) or to 10 nM angiotensin II (AII) was significantly blunted in both parts after preincubation with 1 microM Ch. Depolarization with 25 mM K+ increased [Ca2+]i in both parts. Preincubation with Ch did not affect the increase in [Ca2+]i induced by 25 mM K+. TPA (10 and 100 nM) did not significantly affect the basal [Ca2+]i in the afferent arteriole. The [Ca2+]i response to NA or 25 mM K+ was not affected by TPA. We conclude that blockade of PKC increases [Ca2+]i in afferent arteriolar smooth muscle by a mechanism independent of L-type voltage-sensitive calcium channels. Inhibition of PKC blunts the relative increase in [Ca2+]i in response to AII and, to a lesser extent, that induced by NA. We conclude that PKC might be important in modulating the calcium changes that occur in response to these vasoconstrictors.

Angiotensin II induces a tachyphylactic calcium response in the rabbit afferent arteriole.

The influence of repeated administration of angiotensin II (AII) on smooth muscle calcium concentration ([Ca2+]i) was studied in isolated rabbit renal afferent arterioles loaded with the calcium-sensitive fluorescent probe Fura-2. [Ca2+]i was evaluated in the proximal and distal parts of the afferent arterioles. AII (10(-8) M) increased the [Ca2+]i in both these regions. A second administration of AII, however, did not elicit any response in [Ca2+]i. The response to noradrenaline administration at the end of the experiment was not affected, i.e. there was no fading or cross-desensitization. Since this desensitization was specific for AII, it was of the tachyphylaxis type. Increasing doses of AII (10(-11)-10(-8) M) did not reverse the tachyphylaxis. However, in the proximal part, pretreatment with the voltage-sensitive calcium channel blocker nifedipine (10(-6) M) blunted the tachyphylactic effect of a second administration of AII. When L-arginine (L-Arg) was administered to the bath solution, thus activating the NO system, the development of tachyphylaxis was suppressed in the proximal region. Pretreatment with the protein kinase C (PKC) inhibitor chelerythrine (10(-6) M) did not affect the tachyphylaxis. We conclude that the calcium response to AII in the isolated rabbit afferent arteriole shows tachyphylaxis. This tachyphylaxis cannot be reversed by applying increasing doses of AII (10(-11)-10(-8) M). PKC does not seem to be involved in the tachyphylactic phenomenon in this preparation. It was also found that nifedipine and NO reduced the tachyphylaxis.