1 We have measured the calcium sensitivity in response to noradrenaline stimulation and potassium depolarization of isolated segments of 100 to 200 gm mesenteric resistance vessels from spontaneously hypertensive (SHR) and control Wistar-Kyoto (WKY) rats. The rats were either young (4 wk) or adult (4 months), that is of ages before or after the SHRs had developed elevated blood pressure. Experiments were performed under conditions in which the effect of noradrenaline uptake by, and the potassium-induced noradrenaline release from, the nerve terminals in the vessel walls was eliminated. 2 The response of the SHR and WKY vessels to noradrenaline under conditions where only the extracellular calcium appeared to have been removed was similar. When subsequently stimulated maximally by noradrenaline, the calcium-sensitivity of the SHR vessels (Ca-ED50 -0.1 mM) was greater than that of the WKY vessels (Ca-EDs5 -0.2 mM). When depolarized by potassium, all vessels were less sensitive to calcium and there was little difference in the calcium sensitivity of SHR and WKY vessels in either age group (Ca-ED50 o 0.8 mM). 3 The results suggest that whereas the potassium (potential)-dependent calcium permeability of the SHR smooth muscle cell membrane is normal, the noradrenaline-induced calcium permeability is abnormally high. The presence of this abnormality in the vessels from the young SHRs suggests that it may be a factor involved in the aetiology of hypertension in the SHR.

Liraglutide and exenatide are glucagon-like peptide receptor (GLP-1R) agonists used in the treatment of type 2 diabetes. Both molecules have been associated with the development of thyroid C-cell tumors after lifetime exposure in rodents. Previously, it has been reported that these tumors are preceded by increased plasma calcitonin and C-cell hyperplasia. We can now document that the murine C-cell effects are mediated via GLP-1R. Thus, 13 wk of continuous exposure to GLP-1R agonists was associated with marked increases in plasma calcitonin and in the incidence of C-cell hyperplasia in wild-type mice. In contrast, similar effects were not seen in GLP-1R knockout mice. Human C-cell cancer is often caused by activating mutations in the rearranged-during-transfection (RET) protooncogene. We developed an immunohistochemical method to assess RET activation in tissues. Liraglutide dosing to mice was not found to activate RET. Further evaluation of the signaling pathways demonstrated that liraglutide increased ribosomal S6, but not MAPK kinase, phosphorylation. These observations are consistent with effects of GLP-1R agonists on rodent C cells being mediated via mammalian target of rapamycin activation in a RET- and MAPK-independent manner.

Increased pancreas mass and glucagon-positive adenomas have been suggested to be a risk associated with sitagliptin or exenatide therapy in humans. Novo Nordisk has conducted extensive toxicology studies, including data on pancreas weight and histology, in Cynomolgus monkeys dosed with two different human glucagon-like peptide-1 (GLP-1) receptor agonists. In a 52-week study with liraglutide, a dose-related increase in absolute pancreas weight was observed in female monkeys only. Such dose-related increase was not found in studies of 4, 13, or 87 weeks’ duration. No treatment-related histopathological abnormalities were observed in any of the studies. Quantitative histology of the pancreas from the 52-week study showed an increase in the exocrine cell mass in liraglutide-dosed animals, with normal composition of endocrine and exocrine cellular compartments. Proliferation rate of the exocrine tissue was low and comparable between groups. Endocrine cell mass and proliferation rates were unaltered by liraglutide treatment. Semaglutide showed no increase in pancreas weight and no treatment-related histopathological findings in the pancreas after 13 or 52 weeks’ dosing. Overall, results in 138 nonhuman primates showed no histopathological changes in the pancreas associated with liraglutide or semaglutide, two structurally different GLP-1 receptor agonists.

We have investigated the active, passive and myogenic tension-internal circumference relations of rat intramural coronary and, as controls, mesenteric small arteries (internal diameter ca. 200 micron) using an isometric myograph. The active tensions of the vessels (when fully activated with 30 microM serotonin in K-saline) reached a maximum (2.54 N/m, coronary; 3.39 N/m, mesenteric) at an internal circumference, L0, where the passive tensions (measured in Ca-free solution) were 0.80 N/m (coronary) and 0.74 N/m (mesenteric). Below 0.8 L0 and above 1.2 L0 the active tensions fell linearly, the zero tension intercepts being 0.37 L0 and 1.74 L0 (coronary) and 0.40 L0 and 1.72 L0 (mesenteric). The passive wall tensions of the vessels rose exponentially as a function of internal circumference, the wall tension at 1.5 L0 being 10.0 N/m (coronary) and 8.5 N/m (mesenteric). In normal physiological salt solution, the coronary vessels had a Ca2+ dependent myogenic tone which was also dependent on the internal circumference. Maximum myogenic tone (0.54 N/m) was obtained at 1.18 L0. The mesenteric vessels had no such myogenic tone. Histological examination showed that the media/lumen ratios of both vessel types were the same, and that the smooth muscle content of the media was greater in the coronary (81%) than in the mesenteric (72%) vessels. The smaller active tension of the coronary vessels could not therefore be ascribed to a reduced smooth muscle content, but possibly in part to an observed heterogeneous arrangement of the smooth muscle cells in the coronary vessels.

In this study, two strains of Lactobacillus helveticus were used to produce fermented milk rich in angiotensinconverting enzyme (ACE) inhibitors. In vitro tests revealed that the two milks contained competitive inhibitors of ACE in amounts comparable to what has been obtained in previously reported studies. The two milks were administered by gavage to spontaneously hypertensive rats that had had a permanent aortic catheter inserted through the left arteria carotis, and mean arterial blood pressure and heart rate were monitored from 4 to 8 h after administration. Unfermented milk and milk fermented with a lactococcal strain that does not produce inhibitors were used as controls. Highly significant blood pressure effects were observed; i.e., milk fermented with the two strains of L. helveticus gave a more pronounced drop in blood pressure than the controls. Significant differences in heart rate effects were detected with one of the strains.Angiotensin-converting enzyme (EC 3.4.15.1) (ACE) is known to play a role in the regulation of blood pressure in animals, including humans. The enzyme converts angiotensin I into angiotensin II, the former being an inert peptide and the latter being a pressor agent. The enzyme is also responsible for the breakdown of bradykinin, which is a dilatory peptide. The enzyme is thus an obvious drug target in treatment of certain cardiovascular diseases, including hypertension. Lactic acid bacteria are known to produce inhibitors of the enzyme in various amounts during fermentation (5, 10, 13). The inhibitors are formed by the bacterial proteinases when the lactic acid bacteria hydrolyze milk proteins, mainly casein, into peptides, which can be used as nitrogen sources necessary for growth. All of the ACE inhibitors known to date that are formed in milk during fermentation are peptides that act as competitive inhibitors, such as Ile-Pro-Pro and Val-Pro-Pro (11). In some cases it has been demonstrated that fermented milk rich in inhibitory substances can lower systolic blood pressure in spontaneously hypertensive rats (SHR) after oral administration (11,13,14).Since the inhibitors formed are peptides, which are formed by degradation of caseins, proteolysis plays an important role. In theory, both specificity and overall proteolytic activity may have a significant role in the formation. The fact that the proteolytic systems of lactic acid bacteria are quite unspecific, i.e., that they cleave, for instance, caseins at a high number of places (for a review, see reference 8), combined with the fact that ACE can be inhibited by many different peptidic structures (for instance, Val-Pro-Pro [11], Ile-Tyr [15], and Lys-ValLeu-Pro-Val-Pro [9], all of which inhibit ACE in the micromolar range), led to the investigation of a possible correlation between crude proteolysis (measured as free amino groups) and ACE inhibition. It was shown that, in general, the higher the number of free amino groups formed during fermentation, the higher the chance that the same fermented substance will be able to inhib...