Reference intervals and method sensitivity for electrocardiology, hemodynamics, and body temperature parameters in healthy cynomolgus monkeys.

In nonclinical studies, electrocardiograms (ECG) of cynomolgus monkey are recorded intermittently by external leads in manually restrained animals (snapshot recording) or continuously by jacketed external telemetry (JET) or implanted radiotelemetry transmitter in freely moving animals. With the implanted device, blood pressure and core body temperature can be monitored simultaneously. Despite the frequent use of cynomolgus monkeys in nonclinical safety pharmacology testing, few reference data are available for this species, comparisons of the ECG recording methods are limited, and power analyses are seldom conducted. In this study, pretreatment data were recorded from 406, 663, and 131 healthy experimentally naïve monkeys using the snapshot, JET, and implantable method, respectively, from 2019 to 2021. Reference intervals were determined for ECG, blood pressure, and body temperature parameters. Diurnal effects were observed in these parameters, with the exception of QRS and pulse pressure. The QRS, QT, and heart rate-corrected QTc intervals, as well as blood pressure, had a weak positive relationship with age and/or body weight. There were no sex differences in these parameters, and the country of origin only had minimal influences. Compared to telemetry, snapshot ECG data had shorter RR, PR, and QT intervals and longer QRS interval. The JET and implanted telemetry ECG data were comparable. Effect size analysis was conducted to estimate the method sensitivity for each parameter in common non-clinical study design scenarios. Snapshot recording, JET, and implanted telemetry were sensitive to detect 7-15 milliseconds of changes in QTc intervals in standard study designs, indicating these are powerful methods for assessment of QT prolongation in vivo.

Telemetric assessment of social and single housing: Evaluation of electrocardiographic intervals in jacketed cynomolgus monkeys.

INTRODUCTION: Proactive efforts to socially house laboratory animals are a contemporary, important focus for enhancing animal welfare. Jacketing cynomolgus monkeys has been traditionally considered an exclusionary criterion for social housing based on unsubstantiated concerns that study conduct or telemetry equipment might be compromised. Our objective was to evaluate the effects of jacketing naïve, adolescent cynomolgus monkeys in different single and social housing types based on parallel comparisons of heart rate. METHODS: Eight naive cynomolgus monkeys were randomized into pairs and ECG data were collected for 24h from each animal in each housing condition using a crossover design. Caging paradigms consisted of standard individual, standard pair, quaternary pair (4 linked cages), and European-style pair housing in non-sequential order varied by pair to control for possible time bias. Dosing and blood collection procedures were performed to characterize any effects of housing on ECG data during study conduct. RESULTS: There was no increase in the incidence of equipment damage in pair vs. individually housed animals. Further, animals in all 4 housing paradigms showed similar acclimation assessed as heart rate (mean 139-154 beats per minute), and maintained similar diurnal rhythms, with an expected slowing of the heart rate at night (aggregate lights out HR 110±4bpm compared to daytime 146±7bpm). DISCUSSION: This study demonstrates the effects of different social access and housing types on the study-naïve cynomolgus monkeys during jacketed cardiovascular telemetry data collection in a repeat-dose toxicology study design. There were no discernible effects of social housing on baseline ECG parameters collected via jacketed telemetry, and all animals maintained expected diurnal rhythms in all housing settings tested. These data demonstrate that cynomolgus monkeys can be socially housed during data collection as a standard practice, consistent with global efforts to improve study animal welfare.

Agonist-specific compartmentation of cGMP action in myometrium.

Nitric oxide relaxes myometrium in a cGMP-independent manner. Although cGMP activates its cognate kinase, this is not required for the inhibitory effect of nitric oxide. Thus, nitric oxide-mediated cGMP elevation does not enjoy the same set of substrates as it does in other smooth muscles. To further understand the regulation of relaxation of uterine muscle by cGMP, we have studied the actions of peptide-mediated cGMP action in guinea pig myometrium. We used both functional and biochemical studies of the action of the particulate guanylyl cyclase activator uroguanylin and its receptor, particulate guanylyl cyclase type C, to address the relationship between cGMP elevation acting in the membrane signaling domain to that of the nonmembrane region of the cell. Uroguanylin relaxed oxytocin-induced contractions in a dose-dependent fashion only in pregnant myometrium. Both relaxation and cGMP accumulation after uroguanylin stimulation were blocked by the putative particulate guanylyl cyclase type C inhibitors 2-chloro-ATP and isatin (1H-indole-2,3-dione), but not by the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-A]quinoxalin-1-one (ODQ). Uroguanylin stimulated cGMP accumulation only in the pregnant myometrium. Caveolin-1 expression increased in pregnancy toward term. In the caveolin-1-containing membrane domain, uroguanylin, but not the nitric-oxide donor, led to the elevation of cGMP that was insensitive to ODQ. Particulate guanylyl cyclase C was expressed and prouroguanylin was detected in pregnant myometrium. We conclude that a uroguanylin-particulate cyclase-cGMP relaxation pathway is present and cGMP is compartmented in myometrium. The agonist-mediated selectivity of relaxation to cGMP is of fundamental pharmacological interest in understanding signal transduction in smooth muscle.

Dissociation of cGMP accumulation and relaxation in myometrial smooth muscle: effects of S-nitroso-N-acetylpenicillamine and 3-morpholinosyndonimine.

In guinea pig, primate and man, nitric oxide (NO)-induced regulation of myometrial smooth muscle contraction is distinct from other smooth muscles because cyclic guanosine 3',5'-cyclic monophosphate (cGMP) accumulation is neither necessary nor sufficient to relax the tissue. To further our understanding of the mechanism of action of NO in myometrium, we employed the NO donors, S-nitroso-N-acetylpenicillamine (SNAP), and 3-morpholinosyndonimine (SIN-1) proposed to relax airway smooth muscle by disparate mechanisms involving elevation in intracellular calcium ([Ca(2+)](i)) or cGMP accumulation, respectively. Treatment of guinea pig myometrial smooth muscle with either NO donor at concentrations thought to produce maximal relaxation of smooth muscles resulted in significant elevations in cGMP that were accompanied by phosphorylation of the cGMP-dependent protein kinase substrate vasodilator-stimulated phosphoprotein (VASP), shown here for the first time to be present and phosphorylated in myometrium. Stimulation of myometrial strips with oxytocin (OT, 1 microM) produced an immediate increase in contractile force that persisted in the continued presence of the agonist. Addition of SNAP (100 microM) in the presence of OT relaxed the tissue completely as might be expected of an NO donor. SIN-1 failed to relax the myometrium at any concentration tested up to 300 microM. In Fura-2 loaded myometrial cells prepared from guinea pig, addition of SNAP (100 microM) in the absence of other agonists caused a significant, reproducible elevation of intracellular calcium while SIN-1 employed under the same conditions did not. Our data further support the notion that NO action in myometrium is distinct from that in other smooth muscles and underscores the possibility that discrete regional changes in [Ca(2+)](i), rather than cGMP, signal NO-induced relaxation of the muscle.

Excitatory motor innervation in the canine rectoanal region: role of changing receptor populations.

1. Motor innervation in the canine rectoanal region was examined in isolated strips of the circular muscle layer. Contractile responses to electrical field stimulation began at lower frequencies and were more persistent in the internal anal sphincter (IAS) than in the rectum. 2. Motor innervation to the IAS was almost exclusively sympathetic, since it was blocked by guanethidine (Guan 3 microM) while the response in the proximal rectum was approximately 50% muscarinic, and sensitive to the M(3) selective antagonist 4-diphenylacetoxy-N-methylpiperidine (4-DAMP, 0.1 microM) and 50% tachykinergic, and sensitive to the neurokinin 2 (NK(2)) receptor antagonist GR 94800 (1 microM). From IAS to rectum there was a gradual shift in the relative contribution of intrinsic and extrinsic neural innervation. 3. Responses to exogenously applied transmitters exhibited a similar pattern to that observed with motor innervation. Norepinephrine (NE) was most potent in the IAS and acetylcholine (ACh) and NK-A were most potent in the proximal rectum. The responses were inhibited by prazosin, 4-DAMP and GR 94800 respectively. 4. A gradient in the density of adrenergic alpha(1), muscarinic and NK(2) receptors also existed from IAS to rectum as determined by measuring the binding of [(3)H]-prazosin, [(3)H]-quinuclidinyl benzilate ([(3)H]-QNB and [(3)H]-SR-48968 to smooth muscle membranes. 5. In summary, these data suggest that the shift in motor innervation in the rectoanal region is achieved in part by changes in receptor populations available for activation by sympathetic and enteric motor neurons.