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The second coding exon of the adenosine A, receptor gene was eliminated by homologous recombination. The phenotype of mice (mixed C57B6/129OlaHsd background) was studied, using siblings from matings of heterozygous mice. Among the offspring the ratio between+/+, +/-and -/-animals was 1:2:1. Over the first half-year-at least-growth and viability were the same in all genotypes. Binding of A(1) ligands was eliminated in-/-mice and halved in+/-mice. Blood pressure was increased in-/-mice and this was paralleled by an increase in plasma renin. Heart rate was unaffected, as was contractility. Furthermore, the response of the perfused heart to ischemia was similar in+/+and -/-hearts. However, remote preconditioning was eliminated in-/-mouse hearts. Tubuloglomerular feedback in the kidney was also lost in-/-mice. The analgesic response to a non-selective adenosing receptor agonist was lost in-/-mice, which also showed hyperalgesia in the tail-flick test. There was a slight hypoactivity in-/-mice, but responses to caffeine were essentially normal. The inhibition of excitatory neurotransmission in hippocampus by adenosine was lost in-/-mice and reduced in+/-mice. Responses to ATP were affected similarly. Hypoxic depression of synaptic transmission was essentially eliminated in hippocampus and hypoxic decrease in spinal respiratory neuron firing was markedly reduced. These results show that adenosine A, receptors play a physiologically important role in the kidney, spinal cord, and hippocampus and that they are critically important in the adaptive responses to hypoxia. (C) 2003 Wiley-Liss, Inc. (Less)

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.

The tubuloglomerular feedback (TGF) mechanism is an important regulator of the glomerular filtration rate (GFR) and urine excretion rate. It operates by sensing the distal delivery of fluid at the macula densa site and adjusting the tone of the glomerular arterioles to control GFR. We found evidence that nitric oxide is an important modulator of the setting of the sensitivity of the TGF mechanism. Studies on adenosine A1 receptor deficient mice have shown that these animals lack the TGF response and have an increased renin release. These findings show the important role of adenosine as a mediator of the signal for the TGF mechanism and as an inhibitor of renin release.

Nitric oxide (NO) produced by neuronal NO-synthase (nNOS) in macula densa cells may be involved in the control of renin release. 7-Nitro indazole (7-NI) inhibits nNOS, and we investigated the effect of short- (4 days) and long-term (4 weeks) 7-NI treatment on blood pressure (BP), plasma renin concentration (PRC) and glomerular filtration rate (GFR) in rats on different salt diets. Rats were divided into three groups and given low-salt (LS), normal (C) and high-salt (HS) diets. Each diet group was subdivided into two groups treated either with 7-NI or vehicle. Long-term 7-NI-treated rats (LS and C) showed increased BP compared with controls (LS: 149 +/- 4 vs. 133 +/- 3; C: 146 +/- 4 vs. 127 +/- 4 mmHg). Blood pressure in HS rats did not differ from that in controls. Plasma renin concentration was stimulated in LS-rats (251 +/- 64 mGU mL(-1)) compared with C and HS rats (42 +/- 8 and 39 +/- 5 mGU mL(-1), respectively) but was not significantly affected by chronic 7-NI treatment (350 +/- 103, 49 +/- 10 and 50 +/- 15 mGU mL(-1) in LS, C and HS, respectively). In rats treated with 7-NI for 4 days, no effect on BP was seen, but PRC was increased in 7-NI treated LS rats compared with vehicle treated LS rats (107 +/- 15 vs. 56 +/- 1 mGU mL(-1)). Stimulation of PRC in LS rats was further enhanced by 7-NI after 4 days of treatment, but not affected in rats treated for 4 weeks. This suggests that inhibition of nNOS stimulates renin release but that this stimulatory effect in the long run might be depressed by the increase in blood pressure.

The juxtaglomerular apparatus (JGA) has the very important functions of detecting the fluid flow rate to the distal tubule and thus controlling the glomerular filtration rate (GFR) (tubuloglomerular feedback mechanism [TGF]) and renin release from the afferent arteriole. In studies of the TGF it has been evident that the sensitivity of this mechanism can be reset. Volume expansion will reset it to a low sensitivity leading to a high GFR and urine excretion rate, while dehydration will sensitize the TGF mechanism, giving rise to a low GFR and low urine excretion rate. Furthermore, we have found that in animals that spontaneously develop hypertension there is initially a sensitization of the TGF, leading to a reduced GFR and urine excretion rate, with fluid volume retention in the body and a consequent rise in blood pressure. When the pressure is raised, the TGF characteristics are normalized. In the macula densa (MD) cells in the JGA, there is a large production of NO from neuronal NOS. This production continuously reduces TGF sensitivity and is apparently impaired in animals that spontaneously develop hypertension. When we added an nNOS inhibitor to the drinking water for several weeks while measuring blood pressure, we found an increase in blood pressure after 3-4 weeks of treatment This effect was abolished by a high salt diet From these investigations, it also appeared as if nNOS-derived NO inhibited renin release. Experiments have also indicated that NO may resensitize inhibited G-protein coupled purinergic receptors.

Acute unilateral renal denervation (aDNX) is associated with reduced tubuloglomerular feedback (TGF) sensitivity. Six days after denervation (cDNX) TGF sensitivity is somewhat restored, but TGF reactivity increased. This study aimed to investigate if the increased TGF reactivity that was seen in cDNX kidneys was owing to reduced production of nitric oxide (NO). TGF characteristics were determined with micropuncture experiments in anaesthetized rats, using the stop-flow pressure (PSF) technique. Maximal drop in PSF (DeltaPSF) was used as an index of TGF reactivity and the loop of Henle perfusion rate that elicited half-maximal DeltaPSF, the turning point (TP) was used as a measure of TGF sensitivity. In cDNX kidneys, TP was higher than in control rats (25.4 +/- 1.5 nL min-1 vs. 19.1 +/- 1.1 nL min-1), but clearly lower than in aDNX rats (37. 3 +/- 3.1 nL min-1). TGF was more reactive in cDNX rats (DeltaPSF=14. 7 +/- 1.1 mmHg) than in aDNX (7.9 +/- 1.1 mmHg) and control rats (9. 6 +/- 0.9 mmHg). Intratubular inhibition of NO synthase N omega-nitro-L-arginine (L-NA) in sham-DNX animals, decreased TP to 13.9 +/- 2.2 nL min-1 and DeltaPSF was increased with 92%. In cDNX kidneys TP was not significantly reduced by L-NA, and TGF reactivity was only moderately increased by 31%. Intratubular infusion of L-arginine (L-Arg) reduced DeltaPSF from 10.2 +/- 0.7 to 6.5 +/- 0.6 mmHg in sham-DNX kidneys, but TP was unaffected. In cDNX kidneys, there was no effect on either DeltaPSF or TP by the addition of L-Arg. However, when NO was delivered via sodium nitroprusside in the tubular perfusate, a clear reduction of DeltaPSF was seen in both sham-DNX and cDNX kidneys (from 9.9 +/- 0.5 to 4.4 +/- 1.0 and from14.9 +/- 1.3 to 8.1 +/- 1.5 mmHg, respectively). This indicates that cDNX is a state of low renal NO production and that this low level of NO resets TGF to a higher sensitivity and more pronounced reactivity.