Wild-type Cohort Research Articles

Deficit in the sulfonylurea receptor KATP channel complex compromises the metabolic benefit of exercise training resulting in cardiac deficits

The sulfonylurea-receptor protein that forms the stress-responsive ATP-sensitive potassium (KATP) channel, is the identified binding site for sulfonylurea medications used widely in the treatment of type 2 diabetes mellitus. Clinical studies have indicated a compromised exertional capacity in diabetic patients treated with sulfonylureas yet the mechanism for this drug-induced impairment is unknown. Here the response following genetic ablation of KATP channel function (KO) was compared to wild-type controls following a 28-day endurance swimming protocol. While chronic aquatic training resulted in a lighter, leaner and fitter wild-type cohort, the KO manifested less augmentation in exercise capacity and lacked metabolic improvement in body fat composition and glycemic handling. Skeletal muscle analysis demonstrated myocellular defects compatible with repetitive injury/regeneration. Moreover, the continuous stress of swimming unmasked a survival disadvantage in the KO, associated with pathologic calcium-dependent structural damage in the heart, abnormal hypertrophic development and impaired cardiac function. Thus, intact function of the sulfonylurea-receptor KATP channel complex is mandatory for the attainment of the optimal physiologic benefits of exercise training while preventing injury. This work provides a mechanism for sulfonylurea-cardiovascular exercise intolerance and has wide-ranging implications for diabetic patients treated with KATP channel antagonists. Clinical Pharmacology & Therapeutics (2005) 77, P54–P54; doi: 10.1016/j.clpt.2004.12.097

Phenotype Characterisation Using Integrated Gene Transcript, Protein and Metabolite Profiling

Multifactorial diseases present a significant challenge for functional genomics. Owing to their multiple compartmental effects and complex biomolecular activities, such diseases cannot be adequately characterised by changes in single components, nor can pathophysiological changes be understood by observing gene transcripts alone. Instead, a pattern of subtle changes is observed in multifactorial diseases across multiple tissues and organs with complex associations between corresponding gene, protein and metabolite levels. This article presents methods for exploratory and integrative analysis of pathophysiological changes at the biomolecular level. In particular, novel approaches are introduced for the following challenges: (i) data processing and analysis methods for proteomic and metabolomic data obtained by electrospray ionisation (ESI) liquid chromatography-tandem mass spectrometry (LC/MS); (ii) association analysis of integrated gene, protein and metabolite patterns that are most descriptive of pathophysiological changes; and (iii) interpretation of results obtained from association analyses in the context of known biological processes. These novel approaches are illustrated with the apolipoprotein E3-Leiden transgenic mouse model, a commonly used model of atherosclerosis. We seek to gain insight into the early responses of disease onset and progression by determining and identifying--well in advance of pathogenic manifestations of disease--the sets of gene transcripts, proteins and metabolites, along with their putative relationships in the transgenic model and associated wild-type cohort. Our results corroborate previous findings and extend predictions for three processes in atherosclerosis: aberrant lipid metabolism, inflammation, and tissue development and maintenance.

Cooperation between p53 mutation and high telomerase transgenic expression in spontaneous cancer development.

Telomerase reintroduction in adult somatic tissues is envisioned as a way to extend their proliferative capacity. It is still a question, however, whether constitutive telomerase expression in adult tissues impacts the normal aging and spontaneous cancer incidence of an organism. Here, we studied the aging and spontaneous cancer incidence of mice with transgenic telomerase expression in a wide range of adult tissues, K5-Tert mice. For this, we maintained large colonies of K5-Tert mice for more than 2 years. K5-Tert mice showed a decreased life span compared to wild-type cohorts associated with a higher incidence of preneoplastic and neoplastic lesions in various tissue types. Neoplasias in K5-Tert mice were coincident with transgene expression in the affected tissues. These observations suggest that high telomerase activity may cooperate with genetic alterations that occur with age to promote tumorigenesis. Indeed, we demonstrate here that increased cancer incidence and the reduced viability of K5-Tert mice are aggravated in a p53(+/-) genetic background, indicating that telomerase cooperates with loss of p53 function in inducing tumorigenesis. Altogether, these results demonstrate that constitutive high levels of telomerase activity result in a decreased life span associated with an increased incidence of neoplasias as the organism ages.

Isoflurane and Nociception

The authors recently established that the analgesic actions of the inhalation anesthetic nitrous oxide were mediated by noradrenergic bulbospinal neurons and spinal alpha2B adrenoceptors. They now determined whether noradrenergic brainstem nuclei and descending spinal pathways are responsible for the antinociceptive actions of the inhalation anesthetic isoflurane, and which alpha adrenoceptors mediate this effect. After selective lesioning of noradrenergic nuclei by intracerebroventricular application of the mitochondrial toxin saporin coupled to the antibody directed against dopamine beta hydroxylase (DbetaH-saporin), the antinociceptive action of isoflurane was determined. Antagonists for the alpha1 and alpha2 adrenoceptors were injected at spinal and supraspinal sites in intact and spinally transected rats to identify the noradrenergic pathways mediating isoflurane antinociception. Null mice for each of the three alpha2-adrenoceptor subtypes (alpha2A, alpha2B, and alpha2C) and their wild-type cohorts were tested for their antinociceptive response to isoflurane. Both DbetaH-saporin treatment and chronic spinal transection enhanced the antinociceptive effects of isoflurane. The alpha1-adrenoceptor antagonist prazosin also enhanced isoflurane antinociception at a supraspinal site of action. The alpha2-adrenoceptor antagonist yohimbine inhibited isoflurane antinociception, and this effect was mediated by spinal alpha2 adrenoceptors. Null mice for the alpha2A-adrenoceptor subtype showed a reduced antinociceptive response to isoflurane. The authors suggest that, at clinically effective concentrations, isoflurane can modulate nociception via three different mechanisms: (1) a pronociceptive effect requiring descending spinal pathways, brainstem noradrenergic nuclei, and supraspinal alpha1 adrenoceptors; (2) an antinociceptive effect requiring descending noradrenergic neurons and spinal alpha2A adrenoceptors; and (3) an antinociceptive effect mediated within the spinal cord for which no role for adrenergic mechanism has been found.

Role of bombesin (BN)-like peptides/receptors in emotional behavior by comparison of three strains of BN-like peptide receptor knockout mice

Bombesin (BN)-like peptides are involved in the regulation of a wide variety of behaviors, such as spontaneous activity and feeding. We assessed the role of BN-like peptides/receptors in emotional and/or anxiety-related behavior using three strains of knockout mice, each deficient in a single BN-like peptide receptor (gastrin-releasing peptide receptor, bombesin receptor subtype-3, or neuromedin B receptor). Two representative behavioral paradigms, the light-dark (L-D) box test and the elevated plus maze test, were chosen for this purpose. In these two tests, the level of anxiety can be measured as the preference for exploring the light box, or the length of time spent in the open arms, respectively. By conventional parameters, the only significant finding was that BRS-3-deficient mice exhibited a longer duration of remaining in the open arms compared to the wild-type cohort (P < 0.01). However, analyses of risk assessment behavior revealed that BRS-3-deficient mice exhibited increased 'stretched attend posture' behavior (P < 0.01, compared to wild-type mice in both the L-D box and elevated plus maze tests) while NMB-R-deficient mice exhibited decreased behavior (P < 0.05, compared to wild-type mice in both tests). These results suggest that BN-like peptides/receptors may play a role in modulating emotion including some forms of anxiety (e.g., risk assessment behavior). Further, we found that the type of emotional behavior to which each of the peptide/receptor pathways contributes can be clearly specified.

Mice deficient in oxytocin manifest increased saline consumption following overnight fluid deprivation.

Male mice (9-13 mo of age) in which the gene for oxytocin (OT) had been deleted (OT -/-) were administered 0.5 M sodium chloride (NaCl) solution or tap water as a two-bottle choice test following overnight fluid deprivation (1600 to 1000 the following day). Compared with wild-type cohorts (OT +/+), OT-deficient mice ingested sevenfold greater amounts of saline in the first hour following reintroduction of fluids, P < 0.001, and fourfold greater amounts at the end of 6 h, P < 0.02. No significant difference in total water ingested was noted between the two genotypes at the end of either 1 or 6 h. If food deprivation accompanied the overnight fluid deprivation and food was reintroduced 1 h after the reintroduction of both water and saline, OT -/- mice still ingested greater amounts of saline, but not water, than OT +/+ mice at both 1 h, P < 0.001, and 6 h, P < 0.02. No differences were noted between genotypes in the daily intake of 0.5 M NaCl solution or water during a 3-day observation period before the overnight fluid deprivation. The volume of saline consumed in each 24-h observation period represented about one-tenth of the total fluids ingested in each genotype. We conclude that OT -/- mice display an enhanced salt appetite compared with OT +/+ mice when fluid deprived overnight. The salt appetite was only apparent in the presence of a perturbation such as fluid deprivation, which predisposes the animal to moderate hypovolemia. The observations support an inhibitory role for OT in the control of sodium appetite in mice.

Antinociceptive Action of Nitrous Oxide Is Mediated by Stimulation of Noradrenergic Neurons in the Brainstem and Activation of α2BAdrenoceptors

Although nitrous oxide (N(2)O) has been used to facilitate surgery for >150 years, its molecular mechanism of action is not yet defined. Having established that N(2)O-induced release of norepinephrine mediates the analgesic action at alpha(2) adrenoceptors in the spinal cord, we now investigated whether activation of noradrenergic nuclei in the brainstem is responsible for this analgesic action and which alpha(2) adrenoceptor subtype mediates this property. In rats, Fos immunoreactivity was examined in brainstem noradrenergic nuclei after exposure to nitrous oxide. After selective lesioning of noradrenergic nuclei by intracerebroventricular application of the mitochondrial toxin saporin, coupled to the antibody directed against dopamine beta hydroxylase (DbetaH-saporin), the analgesic and sedative actions of N(2)O were determined. Null mice for each of the three alpha(2) adrenoceptor subtypes (alpha(2A), alpha(2B), and alpha(2C)), and their wild-type cohorts, were tested for their antinociceptive and sedative response to N(2)O. Exposure to N(2)O increased expression of Fos immunoreactivity in each of the pontine noradrenergic nuclei (A5, locus coeruleus, and A7). DbetaH-saporin treatment eliminated nearly all of the catecholamine-containing neurons in the pons and blocked the analgesic but not the sedative effects of N(2)O. Null mice for the alpha(2B) adrenoceptor subtype exhibited a reduced or absent analgesic response to N(2)O, but their sedative response to N(2)O was intact. Our results support a pivotal role for noradrenergic pontine nuclei and alpha(2B) adrenoceptors in the analgesic, but not the sedative effects of N(2)O. Previously we demonstrated that the analgesic actions of alpha(2) adrenoceptor agonists are mediated by the alpha(2A) subtype; taken together with these data we propose that exogenous and endogenous alpha(2) adrenoceptor ligands activate different alpha(2) adrenoceptor subtypes to produce their analgesic action.

Mice deficient in Mac-1 (CD11b/CD18) are less susceptible to cerebral ischemia/reperfusion injury.

Macrophage-1 antigen (Mac-1) (CD11b/CD18), a leukocyte beta2 integrin, facilitates neutrophil adhesion, transendothelial migration, phagocytosis, and respiratory burst, all of which may mediate reperfusion-induced injury to ischemic brain tissue in conditions such as stroke. To determine the role of Mac-1 during ischemia and reperfusion in the brain, we analyzed the effect of transient focal cerebral ischemia in mice genetically engineered with a specific deficiency in Mac-1. Transient focal ischemia/reperfusion was induced by occluding the left middle cerebral artery for 3 hours followed by a 21-hour reperfusion period in Mac-1-deficient (n=12) and wild-type (n=11) mice. Regional cerebral blood flow was determined with a laser-Doppler flowmeter. Brain sections were stained with 2% 2,3,5-triphenyltetrazolium chloride to determine the infarct volume. Neutrophil accumulation was determined by staining the brain sections with dichloroacetate esterase to identify neutrophils. Compared with the wild-type cohort, Mac-1-deficient mice had a 26% reduction in infarction volume (P<0.05). This was associated with a 50%, but statistically insignificant, reduction in the number of extravasated neutrophils in the infarcted areas of the brains in the mutant mice. There were no differences in regional cerebral blood flow between the 2 groups. Mac-1 deficiency reduces neutrophil infiltration and cerebral cell death after transient focal cerebral ischemia. This finding may be related to a reduction in neutrophil extravasation in Mac-1-deficient mice.

Ectopic Expression of Phospholamban in Fast-Twitch Skeletal Muscle Alters Sarcoplasmic Reticulum Ca2+ Transport and Muscle Relaxation

There are three isoforms of the sarcoplasmic reticulum Ca2+-ATPase; they are known as SERCA1, SERCA2, and SERCA3. Phospholamban is present in tissues that express the SERCA2 isoform and is an inhibitor of the affinity of SERCA2 for calcium. In vitro reconstitution and cell culture expression studies have shown that phospholamban can also regulate SERCA1, the fast-twitch skeletal muscle isoform. To determine whether regulation of SERCA1 by phospholamban can be of physiological relevance, we generated transgenic mice that ectopically express phospholamban in fast-twitch skeletal muscle, a tissue normally devoid of phospholamban. Ectopic expression of phospholamban was associated with a decrease in the affinity of SERCA1 for calcium. Assessment of isometric twitch contractions of intact fast-twitch skeletal muscles revealed depressed rates of relaxation in transgenic mice compared with wild-type cohorts. Furthermore, the prolongation of muscle relaxation appeared to correlate with the levels of phospholamban expressed in two transgenic mouse lines. These findings indicate that ectopic expression of phospholamban in fast-twitch skeletal muscle is associated with inhibition of SERCA1 activity and decreased relaxation rates of this muscle.