Increased ATP and ADO Overflow From Sympathetic Nerve Endings and Mesentery Endothelial Cells Plus Reduced Nitric Oxide Are Involved in Diabetic Neurovascular Dysfunction

Abstract

Since the mechanism of human diabetic peripheral neuropathy and vascular disease in type 1 diabetes mellitus remains unknown, we assessed whether sympathetic transmitter overflow is altered by this disease and associated to vascular dysfunction. Diabetes was induced by streptozotocin (STZ)-treatment and compared to vehicle-treated rats. Aliquots of the ex vivo perfused rat arterial mesenteric preparation, denuded of the endothelial layer, were collected to quantify analytically sympathetic nerve co-transmitters overflow secreted by the isolated mesenteries of both groups of rats. Noradrenaline (NA), neuropeptide tyrosine (NPY), and ATP/metabolites were detected before, during, and after electrical field stimulation (EFS, 20 Hz) of the nerve terminals surrounding the mesenteric artery. NA overflow was comparable in both groups; however, basal or EFS-secreted ir-NPY was 26% reduced (p < 0.05) in diabetics. Basal and EFS-evoked ATP and adenosine (ADO) overflow to the arterial mesentery perfusate increased twofold and was longer lasting in diabetics; purine tissue content was 37.8% increased (p < 0.05) in the mesenteries from STZ-treated group of rats. Perfusion of the arterial mesentery vascular territory with 100 μM ATP, 100 nM 2-MeSADP, or 1 μM UTP elicited vasodilator responses of the same magnitude in controls or diabetics, but the increase in luminally accessible NO was 60-70% lower in diabetics (p < 0.05). Moreover, the concentration-response curve elicited by two NO donors was displaced downwards (p < 0.01) in diabetic rats. Parallel studies using primary cultures of endothelial cells from the arterial mesentery vasculature revealed that mechanical stimulation induced a rise in extracellular nucleotides, which in the cells from diabetic rats was larger and longer-lasting when comparing the extracellular release of ATP and ADO values to those of vehicle-treated controls. A 5 min challenge with purinergic agonists elicited a cell media NO rise, which was reduced in the endothelial cells from diabetic rats. Present findings provide neurochemical support for the diabetes-induced neuropathy and show that mesenteric endothelial cells alterations in response to mechanical stimulation are compatible with the endothelial dysfunction related to vascular disease progress.

Keywords: Streptozotocin-induced diabetes; cultured endothelial cells; extracellular adenosine; nitric oxide production; nucleotide release.

FIGURE 1

Time course of noradrenaline (NA) and neuropeptide tyrosine (NPY) released to the rat arterial mesenteric bed perfusates following EFS of sympathetic nerve terminals in endothelium-denuded preparations. Perfusate aliquots of the rat mesentery arterial vascular bed were collected to assay NA (A) and ir-NPY (B) released to the tissue perfusate before, during, and following EFS (symbolized as the 1 min 20 Hz bar). Grey columns indicate vehicle-treated controls (n = 16); open columns correspond to STZ-treated rats (n = 22 for NA determinations, while n = 24 for ir-NPY). Columns indicate the mean average values of the transmitter time course; bars indicate the SEM. ^∗p < 0.05; ^∗∗p < 0.01, is the Mann–Whitney test compared to its matched control at several times.

FIGURE 2

Time course of ATP and related purines detected in the rat arterial mesenteric bed perfusate upon EFS of the tissue nerve terminals in endothelium-denuded preparations. Perfusate aliquots from the rat arterial mesenteric vasculature were collected to assay ATP and related purine metabolites before, during, and after EFS (symbolized as the 1 min 20 Hz stimulation bar) of the tissue’s perivascular nerve terminals. (A) ATP, (B) ADP, (C) AMP, and (D) ADO. Gray columns indicate vehicle-treated controls (n = 10); open columns correspond to STZ-treated rats (n = 19). Columns indicate the mean average values of ATP and purine metabolites along the time course protocol; bars indicate the SEM.^∗p < 0.05; ^∗∗p < 0.01, and ^∗∗∗p < 0.001, in case an ADP, unpaired Student’s t-test was applied, ATP, AMP, and ADO applied Mann–Whitney test as compared to matched control at the several times.

FIGURE 3

Nitric oxide (NO) detection in perfusates of the rat arterial mesenteric vasculature following purinergic agonist administration. Purinergic agonists for several receptor subtypes were perfused to the mesenteric preparation; perfusate aliquots were collected to quantify NO. Perfusion with: (A) ATP, (B) 2-MeSADP, (C) UTP, and (D) BzATP. Results are expressed as Δ NO pmol/mL obtained by subtracting basal values from those elicited following nucleotide application to the mesentery perfusate. Gray columns indicate vehicle-treated controls; open columns correspond to the matched STZ-treated diabetic rats. Columns indicate mean average values; bars represent the S.E.M. Numbers inside the columns represent the rat preparations used in these determinations. ^∗p < 0.05; ^∗∗p < 0.01; in the case of 2-MeSADP, the Mann–Whitney test, ATP, and UTP unpaired Student’s t-test were applied.

FIGURE 4

NO donor concentration-response curves in mesenteries from control and diabetic rats. (A) Representative recordings of 1 μM SNAP-induced vasodilatation in the rat arterial mesenteric bed of a control and a parallel protocol performed on a diabetic rat mesentery. Filled dots represent mesentery pre-contraction with 50 μM NA prior to the addition of NO donors. (B,C) show SNAP and SNP concentration-response curves, respectively, in mesenteries from control and diabetic rats. Symbols indicate mean average values; bars represent the SEM; filled squares refer to controls while the open squares represent the mesenteries from STZ-treated rats. Both the SNAP and SNP curves proved statistical significance when comparing control versus diabetic animals.

FIGURE 5

Time course of extracellular ATP release and its metabolites following cell media displacement from primary rat mesentery ECs culture. Cell media aliquots from wells not exposed to CMD (basal values), or parallel aliquots from wells 1, 3, 5, and 10 min after CMD, were collected from vehicle-treated controls (gray columns) or ECs derived from mesenteries of STZ-treated animals (open columns). (A) ATP, (B) ADP, (C) AMP, and (D) ADO. Columns indicate mean average values; bars refer to the SEM. Numbers at the bottom of each column indicate replicas of each purine detection assay. ^∗p < 0.05; ^∗∗p < 0.01, ^∗∗∗p < 0.001, compare the release of these purines in cell cultures from controls and STZ-treated rats. ATP and ADO values were compared using the Mann–Whitney test, while for the ADP comparisons the unpaired Student’s t-test was used.