These data indicate that diabetes increases BBB permeability via a loss of tight junction proteins, and that increased BBB permeability in diabetes does not result from hyperglycaemia alone. Increased plasma MMP activity is implicated in degradation of BBB tight junction proteins and increased BBB permeability in diabetes. Peripheral MMP activity may present a novel target for protection of the BBB and prevention of neurological complications in diabetes.

Aquaporin-1 (AQP1) is a member of the membrane intrinsic protein (MIP) gene family and is known to provide pathways for water flux across cell membranes. We show here that cloned human AQP1 not only mediates water flux but also serves as a cGMP-gated ion channel. Two-electrode voltage-clamp analyses showed consistent activation of an ionic conductance in wild-type AQP1-expressing oocytes after the direct injection of cGMP (50 nl of 100 mM). Current activation was not observed in control (water-injected) oocytes or in AQP5-expressing oocytes with osmotic water permeabilities equivalent to those seen with AQP1. Patch-clamp recordings revealed large conductance channels (150 pS in K(+) saline) in excised patches from AQP1-expressing oocytes after the application of cGMP to the internal side. Amino acid sequence alignments between AQP1 and sensory cyclic-nucleotide-gated channels showed similarities between the cyclic-nucleotide-gated binding domain and the AQP1 carboxyl terminus that were not present in AQP5. Competitive radioligand-binding assays with [(3)H]cGMP demonstrated specific binding (K(D) = 0.2 microM) in AQP1-expressing Sf9 cells but not in controls. These results indicate that AQP1 channels have the capacity to participate in ionic signaling after the activation of cGMP second-messenger pathways.

Epithelial sodium channel (ENaC) subunit (alpha, beta, and gamma) mRNA and protein have been localized to the principal cells of the connecting tubule (CNT), cortical collecting duct (CCD), and outer medullary collecting duct (OMCD) in rat kidney. However, the subcellular localization of ENaC subunits in the principal cells of these cells is undefined. The cellular and subcellular localization of ENaC subunits in rat kidney was therefore examined. Immunocytochemistry demonstrated the presence of all three subunits in principal cells of the CNT, CCD, OMCD, and IMCD. In cortex and outer medulla, confocal microscopy demonstrated a difference in the subcellular localization of subunits. alpha-ENaC was localized mainly in a zone in the apical domains, whereas beta- and gamma-ENaC were found throughout the cytoplasm. Immunoelectron microscopy confirmed the presence of ENaC subunits in both the apical plasma membrane and intracellular vesicles. In contrast to the labeling pattern seen in cortex, alpha-ENaC labeling in IMCD cells was distributed throughout the cytoplasm. In the urothelium covering pelvis, ureters, and bladder, immunoperoxidase and confocal microscopy revealed differences the presence of all ENaC subunits. As seen in CCD, alpha-ENaC was present in a narrow zone near the apical plasma membrane, whereas beta- and gamma-ENaC were dispersed throughout the cytoplasm. In conclusion, all three subunits of ENaC are expressed throughout the collecting duct (CD), including the IMCD as well as in the urothelium. The intracellular vesicular pool in CD principal cells suggests ENaC trafficking as a potential mechanism for the regulation of Na(+) reabsorption.

Abstract-We carried out semiquantitative immunoblotting of kidney to identify apical sodium transporter proteins whose abundances are regulated by angiotensin II. In NaCl-restricted rats (0.5 mEq Na/200 g BW/d), the type 1 angiotensin II receptor (AT 1 receptor) antagonist, candesartan, (1 mg/kg of body weight per day SC for 2 days) markedly decreased the abundance of the ␣ subunit of the epithelial sodium channel (ENaC). This subunit has been shown to be rate-limiting for assembly of mature ENaC complexes. In addition, systemic infusion of angiotensin II increased ␣ENaC protein abundance in rat kidney cortex. The decrease in ␣ENaC protein abundance in response to AT 1 receptor blockade was associated with a fall in ␣ENaC mRNA abundance (real-time RT-PCR), consistent with transcriptionally mediated regulation. The effect of AT 1 receptor blockade on ␣ENaC expression was not blocked by spironolactone, suggesting a direct role of the AT 1 receptor in regulation of ␣ENaC gene expression. Candesartan administration was also found to increase the abundances of the ␤ and ␥ subunits. The increase in ␤ and ␥ENaC protein abundance was not associated with a significant increase in the renal abundances of the corresponding mRNAs, suggesting a posttranscriptional mechanism. Immunocytochemistry confirmed the increase in ␤ and ␥ENaC protein abundance and demonstrated candesartan-induced ENaC internalization in collecting duct cells. The results support the view that the angiotensin II receptor regulates ENaC abundance, consistent with a role for angiotensin II in regulation of collecting duct function. Key Words: receptors, angiotensin II Ⅲ angiotensin antagonist Ⅲ sodium channels Ⅲ aldosterone L ong-term control of blood pressure is closely tied to sodium balance and extracellular fluid volume regulation, both of which are controlled in part by the renin-angiotensin-aldosterone system (RAAS). 1 Angiotensin II has important nonrenal effects that are instrumental in the control of blood pressure as both a vasoconstrictor and a regulator of aldosterone secretion. In addition, angiotensin II has direct effects on the renal tubule in regulating NaCl reabsorption. 2 The direct antinatriuretic effects of angiotensin II appear to be particularly important in conditions of dietary sodium restriction or contraction of extracellular fluid volume. 1 Regulation of renal tubule sodium transport by angiotensin II has been investigated chiefly in relatively short-term experiments with observations within a few minutes of angiotensin II addition. 3-7 However, there is growing evidence that a variety of mediators of transport regulation in the kidney, such as vasopressin 8 and aldosterone, 9 work by both short-term and long-term actions. The long-term actions are associated with adaptive increases in abundance of transporter proteins, whereas short-term actions are generally associated with regulated trafficking or posttranslational modifications of the transporter proteins.The antinatriuretic effects of angiotensin II on sodium transport are...

The brain contains a subpopulation of glucosensing neurons that alter their firing rate in response to elevated glucose concentrations. In pancreatic ␤-cells, glucokinase (GK), the rate-limiting enzyme in glycolysis, mediates glucose-induced insulin release by regulating intracellular ATP production. A similar role for GK is proposed to underlie neuronal glucosensing. Via in situ hybridization, GK mRNA was localized to hypothalamic areas that are thought to contain relatively large populations of glucosensing neurons (the arcuate, ventromedial, dorsomedial, and paraventricular nuclei and the lateral area). GK also was found in brain areas without known glucosensing neurons (the lateral habenula, the bed nucleus stria terminalis, the inferior olive, the retrochiasmatic and medial preoptic areas, and the thalamic posterior paraventricular, interpeduncular, oculomotor, and anterior olfactory nuclei). Conversely, GK message was not found in the nucleus tractus solitarius, which contains glucosensing neurons, or in ependymal cells lining the third ventricle, where others have described its presence. In the arcuate nucleus, >75% of neuropeptide Y-positive neurons also expressed GK, and most GK + neurons also expressed KIR6.2 (the pore-forming subunit of the ATP-sensitive K + channel). The anatomic distribution of GK mRNA was confirmed in micropunch samples of hypothalamus via reverse transcription-polymerase chain reaction (RT-PCR). Nucleotide sequencing of the recovered PCR product indicated identity with nucleotides 1092-1411 (within exon 9 and 10) of hepatic and ␤-cell GK. The specific anatomic localization of GK mRNA in hypothalamic areas known to contain glucosensing neurons and the coexpression of KIR6.2 and NPY in GK + neurons support a role for GK as a primary determinant of glucosensing in neuropeptide neurons that integrate multiple signals relating to peripheral energy metabolism. Diabetes 49:693-700, 2000 M ammalian feeding behavior and general energy homeostasis appear to be regulated by circulating levels of nutrients (glucose) and peptides (e.g., leptin, insulin). Sensors to detect levels of these factors have been found to reside within specific nuclei of the hypothalamus (1-8), where central regulation of energy homeostasis is believed to be coordinated. For example, large changes in blood glucose are correlated with centrally mediated responses such as thermogenesis through activation of the sympathetic nervous system. These changes are monitored by the brain (9-11), and such responses are altered in obesity-prone animals (11-13). Moreover, lesions of the ventromedial hypothalamus (VMH) prevent the hypoglycemic activation of the sympathetic response (14). Thus, available data indicate that glucose detection by hypothalamic neurons may play an important role in regulating energy homeostasis.Glucosensing neurons are among the best characterized of such metabolic sensors. Unlike most neurons, they use glucose as a signaling molecule to alter their firing rate in response to changes in ambient glucose le...

There is extensive evidence that activation of the immune system is both necessary and required for the development of Ang II-induced hypertension in males. The purpose of this study was to determine if sex differences exist in the ability of the adaptive immune system to induce Ang II-dependent hypertension and whether central and renal T cell infiltration during Ang II-induced hypertension is sex-dependent. Rag-1−/− mice, lacking both T and B cells, were used. Male and female Rag-1−/− mice received adoptive transfer of male CD3+ T cells 3 weeks prior to 14 day Ang II infusion (490ng/kg/min). Blood pressure was monitored via tail cuff. In the absence of T cells, systolic blood pressure (SBP) responses to Ang II were similar between sexes (Δ22.1mmHg males vs. Δ18mmHg females). After adoptive transfer of male T cells, Ang II significantly increased SBP in males (Δ37.7mmHg, p<0.05) compared to females (Δ13.7mmHg). Flow cytometric analysis of total T cells and CD4+, CD8+, and regulatory Foxp3+-CD4+ T cell subsets identified that renal lymphocyte infiltration was significantly increased in males vs females in both control and Ang II infused animals (p<0.05). Immunohistochemical staining for CD3+ positive T cells in the SFO region of the brain was increased in males compared to females. These results suggest that female Rag-1−/− mice are protected from male T cell-mediated increases in Ang II-induced hypertension as compared to their male counterparts, and this protection may involve sex differences in the magnitude of T cell infiltration of the kidney and brain.

We have used peptide-directed antibodies to each major renal Na transporter and channel proteins to screen renal homogenates for changes in Na transporter protein expression after initiation of dietary NaCl restriction. After equilibration on a NaCl-replete diet (2.0 meq · 200 g body wt−1 · day−1), rats were switched to a NaCl-deficient diet (0.02 meq · 200 g body wt−1 · day−1). Na excretion fell to 25% of baseline levels on day 1, followed by a further decrease <4% of baseline levels on day 3, of NaCl restriction. The decreased Na excretion at day 1 occurred despite the absence of a significant increase in plasma aldosterone level or in the abundance of any of the major renal Na transporters. However, after a 1-day lag, plasma aldosterone levels increased in association with increases in abundances of three aldosterone-regulated Na transporter proteins: the thiazide-sensitive Na-Cl cotransporter (NCC), the α-subunit of the amiloride-sensitive epithelial Na channel (α-ENaC), and the 70-kDa form of γ-ENaC. RNase protection assays of transporter mRNA levels revealed an increase in renal α-ENaC mRNA coincident with the increase in α-ENaC protein abundance. However, there was no change in NCC mRNA abundance, suggesting that the increase in NCC protein in response to dietary NaCl restriction was not a result of altered gene transcription. These results point to early regulatory processes that decrease renal Na excretion without an increase in the abundance of any Na transporter, followed by a late aldosterone-dependent response associated with upregulation of NCC and ENaC.

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