Reduction in Hindquarter Vascular Resistance Supports 5-HT7 Receptor Mediated Hypotension.

The 5-HT7 receptor is the primary mediator of both the acute (

Activation of the 5-HT7 receptor but not nitric oxide synthase is necessary for chronic 5-hydroxytryptamine-induced hypotension.

NEW FINDINGS: What is the central question of this study? What mechanisms account for the hypotension observed during chronic elevations in circulating 5-hydroxytryptamine in rats? What is the main finding and its importance? Chronic 5-hydroxytryptamine-induced hypotension requires continued activation of the 5-HT7 receptor subtype but does not require NO, an outcome that resolves previous conflicting results. Therapeutic interruption of the hypotensive actions of 5-HT under pathophysiological conditions can only be achieved through blockade of the 5-HT7 receptor. ABSTRACT: Low dose infusion of 5-hydroxytryptamine (5-HT) to rats causes both an acute and a chronic fall in arterial blood pressure. The 5-HT7 receptor subtype plays a critical part in the observed hypotension. Acute (minutes to hours) 5-HT infusion shows no depressor role for nitric oxide (NO), but 5-HT depressor responses under chronic conditions suggest that NO production may be critical. We test the hypothesis that NO contributes to the chronic, but not the acute, depressor response to 5-HT. We compared the role of NO and 5-HT7 receptors in 5-HT-induced hypotension under acute and chronic conditions in the same animal. Mean arterial pressure and heart rate were measured by radiotelemetry in conscious rats during 5 days of saline or 5-HT (25 µg kg-1  min-1 ; osmotic pump) infusion and for 2 days after infusion was stopped. To quantify the contributions of NO and the 5-HT7 receptor to 5-HT-induced hypotension, the nitric oxide synthase (NOS) inhibitor l-NAME or the selective 5-HT7 receptor antagonist SB-267790 were given at 1, 3 and 5 days of chronic infusion, and 1 day after 5-HT infusion pumps were removed. Nω -Nitro-l-arginine methyl ester (l-NAME) caused a pressor response of the same magnitude in the absence or presence of 5-HT infusion. Conversely, SB-269970 did not affect mean arterial pressure in the absence of 5-HT infusion and reversed the 5-HT-induced depressor response at each time point. Our findings demonstrate that acute and chronic 5-HT-induced hypotension does not require NOS activation but does require continued activation of the 5-HT7 receptor.

Creation of the 5-hydroxytryptamine receptor 7 knockout rat as a tool for cardiovascular research.

Using CRISPR-Cas9 technology, we created a 5-HT7 receptor global knockout (KO) rat, on a Sprague-Dawley background, for use in cardiovascular physiology studies focused on blood pressure regulation. A stable line carrying indels in exons 1 and 2 of the rat Htr7 locus was established and validated. Surprisingly, 5-HT7 receptor mRNA was still present in the KO rat. However, extensive cDNA and genomic sequencing of KO tissues confirmed an 11 bp deletion in exon 1 and 4 bp deletion in exon 2. The exon 1 deletion resulted in a frameshifted mRNA sequence coding for a nonfunctional protein. While the Htr1B locus was a potential off-target for the guide RNAs designed for exon 2 of Htr7, there were no off-target sequence changes at this locus in the originating founder. When the F2 generation of KO was compared with wild-type (WT) counterparts, neither the male nor female KO rats were different in body size, fat weights, or mass of organs (kidney, heart, and brain) important to blood pressure. Females were smaller in mass than their counterpart males. Clinical measures of plasma from nonfasted rats revealed largely similar values, comparing WT and KO, of glucose, blood urea nitrogen, creatinine, phosphate, calcium, and albumin to name a few. Loss of a functional 5-HT7 receptor was validated by the complete loss of relaxation to the 5-HT1/7 receptor agonist 5-carboxamidotryptamine in the isolated abdominal vena cava. This newly created 5-HT7 receptor KO rat will be of use to investigate the importance of the 5-HT7 receptor in blood pressure regulation.

5-HT does not lower blood pressure in the 5-HT7 knockout rat.

The fall in mean arterial pressure (MAP) after 24 h of 5-HT infusion is associated with a dilation of the portal vein (PV) and abdominal inferior vena cava (Ab IVC); all events were blocked by the selective 5-HT7 receptor antagonist SB269970. Few studies have investigated the contribution of the 5-HT7 receptor in long-term cardiovascular control, and this requires an understanding of the chronic activation of the receptor. Using the newly created 5-HT7 receptor knockout (KO) rat, we presently test the hypothesis that continuous activation of the 5-HT7 receptor by 5-HT is necessary for the chronic (1 wk) depressor response and splanchnic venodilation. We also address if the 5-HT7 receptor contributes to endogenous cardiovascular regulation. Conscious MAP (radiotelemeter), splanchnic vessel diameter (ultrasound), and cardiac function (echocardiogram) were measured in ambulatory rats during multiday 5-HT infusion (25 µg·kg-1·min-1 via minipump) and after pump removal. 5-HT infusion reduced MAP and caused splanchnic venodilation of wild-type (WT) but not KO rats at any time point. The efficacy of 5-HT-induced contraction was elevated in the isolated abdominal inferior vena cava from the KO compared with WT rats, supporting loss of a relaxant receptor. Similarly, the efficacy of 5-HT causing an acute pressor response to higher doses of 5-HT in vivo was also increased in the KO vs. WT rat. Our work supports a novel mechanism for the cardiovascular effects of 5-HT, activation of 5-HT7 receptors mediating venodilation in the splanchnic circulation, which could prove useful in the treatment of cardiovascular disease.

Whole-Body but Not Hepatic Knockdown of Chemerin by Antisense Oligonucleotide Decreases Blood Pressure in Rats.

Chemerin is an inflammatory adipokine positively associated with hypertension and obesity. The majority of chemerin derives from the liver and adipose tissue, however, their individual contributions to blood pressure are unknown. We began studying chemerin in the normal rat using antisense oligonucleotides (ASO) with whole-body activity (Gen 2.5 chemerin ASO) or liver-restricted activity (GalNAc chemerin ASO). We hypothesized that in normotensive male Sprague-Dawley rats, circulating chemerin is predominately liver-derived and regulates blood pressure. A dosing study of the Gen 2.5 chemerin ASO (with a scrambled control ASO) supported 25 mg/kg as the appropriate dose. GalNAc chemerin ASO was also assessed and used at 10 mg/kg. Radiotelemetry monitored mean arterial pressure (MAP) for a 1-week baseline and weekly subcutaneous ASO injections for 4 weeks. Two days after the final injection, animals were euthanized for tissue reverse transcription-polymerase chain reaction and chemerin Western analysis. Gen 2.5 chemerin ASO treatments reduced chemerin mRNA and protein in liver, retroperitoneal fat (RP), and mesenteric perivascular adipose tissue (mPVAT), as well as reducing protein in plasma. GalNAc chemerin ASO treatments reduced chemerin mRNA and protein in liver and chemerin protein in plasma but had no effect on expression in RP fat or mPVAT. Gen 2.5 chemerin ASO treatment reduced MAP compared with control ASO but was unchanged in animals receiving the GalNAc chemerin ASO. Although circulating chemerin is liver-derived, it does not play a major role in blood pressure regulation. Local effects of chemerin from fat may explain this discrepancy and support chemerin's association with hypertension and obesity.

5-HT causes splanchnic venodilation.

Serotonin [5-hydroxytryptamine (5-HT)] causes relaxation of the isolated superior mesenteric vein, a splanchnic blood vessel, through activation of the 5-HT7 receptor. As part of studies designed to identify the mechanism(s) through which chronic (≥24 h) infusion of 5-HT lowers blood pressure, we tested the hypothesis that 5-HT causes in vitro and in vivo splanchnic venodilation that is 5-HT7 receptor dependent. In tissue baths for measurement of isometric contraction, the portal vein and abdominal inferior vena cava relaxed to 5-HT and the 5-HT1/7 receptor agonist 5-carboxamidotryptamine; relaxation was abolished by the 5-HT7 receptor antagonist SB-269970. Western blot analyses showed that the abdominal inferior vena cava and portal vein express 5-HT7 receptor protein. In contrast, the thoracic vena cava, outside the splanchnic circulation, did not relax to serotonergic agonists and exhibited minimal expression of the 5-HT7 receptor. Male Sprague-Dawley rats with chronically implanted radiotelemetry transmitters underwent repeated ultrasound imaging of abdominal vessels. After baseline imaging, minipumps containing vehicle (saline) or 5-HT (25 µg·kg-1·min-1) were implanted. Twenty-four hours later, venous diameters were increased in rats with 5-HT-infusion (percent increase from baseline: superior mesenteric vein, 17.5 ± 1.9; portal vein, 17.7 ± 1.8; and abdominal inferior vena cava, 46.9 ± 8.0) while arterial pressure was decreased (~13 mmHg). Measures returned to baseline after infusion termination. In a separate group of animals, treatment with SB-269970 (3 mg/kg iv) prevented the splanchnic venodilation and fall in blood pressure during 24 h of 5-HT infusion. Thus, 5-HT causes 5-HT7 receptor-dependent splanchnic venous dilation associated with a fall in blood pressure.NEW & NOTEWORTHY This research is noteworthy because it combines and links, through the 5-HT7 receptor, an in vitro observation (venorelaxation) with in vivo events (venodilation and fall in blood pressure). This supports the idea that splanchnic venodilation plays a role in blood pressure regulation.

Serial Measurements of Splanchnic Vein Diameters in Rats Using High-Frequency Ultrasound.

The purpose of this study was to investigate serial ultrasound imaging in rats as a fully non-invasive method to (1) quantify the diameters of splanchnic veins in real time as an indirect surrogate for the capacitance function of those veins, and (2) assess the effects of drugs on venous dimensions. A 21 MHz probe was used on anesthetized male Sprague-Dawley rats to collect images containing the portal vein (PV), superior mesenteric vein (SMV), abdominal inferior vena cava (IVC), and splenic vein (SpV; used as a landmark in timed studies) and the abdominal aorta (AA). Stable landmarks were established that allowed reproducible quantification of cross-sectional diameters within an animal. The average diameters of vessels measured every 5 min over 45 min remained within 0.75 ± 0.15% (PV), 0.2 ± 0.09% (SMV), 0.5 ± 0.12% (IVC), and 0.38 ± 0.06% (AA) of baseline (PV: 2.0 ± 0.12 mm; SMV: 1.7 ± 0.04 mm; IVC: 3.2 ± 0.1 mm; AA: 2.3 ± 0.14 mm). The maximal effects of the vasodilator sodium nitroprusside (SNP; 2 mg/kg, i.v. bolus) on venous diameters were determined 5 min post SNP bolus; the diameters of all noted veins were significantly increased by SNP, while mean arterial pressure (MAP) decreased 29 ± 4 mmHg. By contrast, administration of the venoconstrictor sarafotoxin (S6c; 5 ng/kg, i.v. bolus) significantly decreased PV and SpV, but not IVC, SMV, or AA, diameters 5 min post S6c bolus; MAP increased by 6 ± 2 mmHg. In order to determine if resting splanchnic vein diameters were stable over much longer periods of time, vessel diameters were measured every 2 weeks for 8 weeks. Measurements were found to be highly reproducible within animals over this time period. Finally, to evaluate the utility of vein imaging in a chronic condition, images were acquired from 4-week deoxycorticosterone acetate salt (DOCA-salt) hypertensive and normotensive (SHAM) control rats. All vessel diameters increased from baseline while MAP increased (67 ± 4 mmHg) in DOCA-salt rats compared to SHAM at 4 weeks after pellet implantation. Vessel diameters remained unchanged in SHAM animals. Together, these results support serial ultrasound imaging as a non-invasive, reliable technique able to measure acute and chronic changes in the diameter of splanchnic veins in intact rats.

5-Hydroxytryptamine does not reduce sympathetic nerve activity or neuroeffector function in the splanchnic circulation.

Infusion of 5-hydroxytryptamine (5-HT) in conscious rats results in a sustained (up to 30 days) fall in blood pressure. This is accompanied by an increase in splanchnic blood flow. Because the splanchnic circulation is regulated by the sympathetic nervous system, we hypothesized that 5-HT would: 1) directly reduce sympathetic nerve activity in the splanchnic region; and/or 2) inhibit sympathetic neuroeffector function in splanchnic blood vessels. Moreover, removal of the sympathetic innervation of the splanchnic circulation (celiac ganglionectomy) would reduce 5-HT-induced hypotension. In anaesthetized Sprague-Dawley rats, mean blood pressure was reduced from 101±4 to 63±3mm Hg during slow infusion of 5-HT (25µg/kg/min, i.v.). Pre- and postganglionic splanchnic sympathetic nerve activity were unaffected during 5-HT infusion. In superior mesenteric arterial rings prepared for electrical field stimulation, neither 5-HT (3, 10, 30nM), the 5-HT1B receptor agonist CP 93129 nor 5-HT1/7 receptor agonist 5-carboxamidotryptamine inhibited neurogenic contraction compared to vehicle. 5-HT did not inhibit neurogenic contraction in superior mesenteric venous rings. Finally, celiac ganglionectomy did not modify the magnitude of fall or time course of 5-HT-induced hypotension when compared to animals receiving sham ganglionectomy. We conclude it is unlikely 5-HT interacts with the sympathetic nervous system at the level of the splanchnic preganglionic or postganglionic nerve, as well as at the neuroeffector junction, to reduce blood pressure. These important studies allow us to rule out a direct interaction of 5-HT with the splanchnic sympathetic nervous system as a cause of the 5-HT-induced fall in blood pressure.

5-HT is a potent relaxant in rat superior mesenteric veins.

Serotonin (5-HT, 5-hydroxytryptamine) reduces blood pressure of the conscious rat when administered chronically (1 week). 5-HT does not directly relax isolated arteries, and microsphere experiments in 5-HT-infused rats suggested that 5-HT increased flow to the splanchnic bed. We hypothesized that 5-HT increased splanchnic flow because of direct venous relaxation; our focus was thus on the superior mesenteric vein (SMV) as an important vein in splanchnic circulation. Real-time RT-PCR, immunohistochemistry and Western analyses supported the predominant expression of the 5-HT2B and 5-HT7 receptor in the SMV. The SMV was mounted in tissue baths for measurement of isometric contraction. 5-HT caused a concentration-dependent relaxation of the endothelin-1 (ET-1)-contracted vein. The threshold of 5-HT-induced venous relaxation was significantly lower than for 5-HT-induced venous contraction (∼2 vs. 700 nmol/L, respectively). A series of serotonergic agonists established in their use of receptor characterization was tested, and the following rank order of potency found for agonist-induced relaxation (receptor selectivity): 5-CT (5-HT1/5-HT7)>5-HT = LP-44 (5-HT7)>PNU109291 (5-HT1D) = BW723C86 (5-HT2B). 8-OH-DPAT (5-HT1A/7), CP93129 (5-HT1B), mCPBG (5-HT3/4), AS19 (5-HT7) and TCB-2 (5-HT2A) did not relax the isolated vein. Consistent with these findings, two different 5-HT7 receptor antagonists SB 269970 and LY215840 but not the 5-HT2B receptor antagonist LY272015 nor the nitric oxide synthase inhibitor LNNA abolished 5-CT-induced relaxation of the isolated SMV. 5-CT (1 µg kg(-1) min(-1), sc) also reduced blood pressure over 7 days. These findings suggest that 5-HT directly relaxes the SMV primarily through activation of the 5-HT7 receptor.

Chemerin connects fat to arterial contraction.

OBJECTIVE: Obesity and hypertension are comorbid in epidemic proportion, yet their biological connection is largely a mystery. The peptide chemerin is a candidate for connecting fat deposits around the blood vessel (perivascular adipose tissue) to arterial contraction. We presently tested the hypothesis that chemerin is expressed in perivascular adipose tissue and is vasoactive, supporting the existence of a chemerin axis in the vasculature. APPROACH AND RESULTS: Real-time polymerase chain reaction, immunohistochemistry, and Western analyses supported the synthesis and expression of chemerin in perivascular adipose tissue, whereas the primary chemerin receptor ChemR23 was expressed both in the tunica media and endothelial layer. The ChemR23 agonist chemerin-9 caused receptor, concentration-dependent contraction in the isolated rat thoracic aorta, superior mesenteric artery, and mesenteric resistance artery, and contraction was significantly amplified (more than 100%) when nitric oxide synthase was inhibited and the endothelial cell mechanically removed or tone was placed on the arteries. The novel ChemR23 antagonist CCX832 inhibited phenylephrine-induced and prostaglandin F2α-induced contraction (+perivascular adipose tissue), suggesting that endogenous chemerin contributes to contraction. Arteries from animals with dysfunctional endothelium (obese or hypertensive) demonstrated a pronounced contraction to chemerin-9. Finally, mesenteric arteries from obese humans demonstrate amplified contraction to chemerin-9. CONCLUSIONS: These data support a new role for chemerin as an endogenous vasoconstrictor that operates through a receptor typically attributed to function only in immune cells.

Smooth muscle pharmacology in the isolated virgin and pregnant rat uterus and cervix.

Uterine smooth muscle function is established, but comparatively little is known about cervical smooth muscle pharmacology. We performed a proof-of-principle experiment that smooth muscle was expressed in the cervix in both virgin and pregnant rats, using the uterus as a comparator. We tested whether all tissues were pharmacologically responsive to contractile and relaxant agonists. Immunohistochemistry revealed the expression of smooth muscle α-actin in all tissues. The isolated tissue bath was used to measure isometric contractility of uterine strips and whole cervices from virgin and pregnant (day 11 ± 2) female Sprague-Dawley rats. We tested classic activators of uterine smooth muscle contraction and relaxation in both uterus and cervix. All tissues contracted to the depolarizing agent potassium chloride, prostaglandin F2α, muscarinic cholinergic agonist carbachol [2-[(aminocarbonxyl)oxy]-N,N,N-trimethylethanaminium chloride], and 5-hydroxytryptamine. Unlike other tissues, the pregnant cervix did not contract to oxytocin, but the oxytocin receptor was present. Both cervix and uterus (virgin and pregnant) had concentration-dependent, near-complete relaxation to the adrenergic agonist norepinephrine and adenylate cyclase activator forskolin [(3R,4aR,5S,6S,6aS,10S,10aR,10bS)-6,10-10b-trihydroxy-3,4a,7,10a-pentamethyl-1-oxo-3-vinyldodecahydro-1H-benzo[f] chroment-5-yl acetate]. The ß-adrenergic receptor agonist isoproterenol was less potent in pregnant cervix versus virgin by ∼10-fold. All tissues, particularly the cervix, responded poorly to the nitric-oxide donor sodium nitroprusside, relaxing ∼20% maximally. These findings support the importance of smooth muscle in the cervix, the use of the isolated cervix in pharmacological studies, and a similarity between smooth muscle pharmacology of the nonpregnant uterus and cervix. This work highlights the unappreciated smooth muscle function of the cervix versus uterus and cervical changes in pharmacology during pregnancy.