GPR55, a G-Protein Coupled Receptor for Lysophosphatidylinositol,Plays a Role in Motor Coordination

The G-protein coupled receptor 55 (GPR55) is activated by lysophosphatidylinositols and some cannabinoids. Recent studies found prominent roles for GPR55 in neuropathic/inflammatory pain, cancer and bone physiology. However, little is known about the role of GPR55 in CNS development and function. To address this question, we performed a detailed characterization of GPR55 knockout mice using molecular, anatomical, electrophysiological, and behavioral assays. Quantitative PCR studies found that GPR55 mRNA was expressed (in order of decreasing abundance) in the striatum, hippocampus, forebrain, cortex, and cerebellum. GPR55 deficiency did not affect the concentrations of endocannabinoids and related lipids or mRNA levels for several components of the endocannabinoid system in the hippocampus. Normal synaptic transmission and short-term as well as long-term synaptic plasticity were found in GPR55 knockout CA1 pyramidal neurons. Deleting GPR55 function did not affect behavioral assays assessing muscle strength, gross motor skills, sensory-motor integration, motor learning, anxiety or depressive behaviors. In addition, GPR55 null mutant mice exhibited normal contextual and auditory-cue conditioned fear learning and memory in a Pavlovian conditioned fear test. In contrast, when presented with tasks requiring more challenging motor responses, GPR55 knockout mice showed impaired movement coordination. Taken together, these results suggest that GPR55 plays a role in motor coordination, but does not strongly regulate CNS development, gross motor movement or several types of learned behavior.     

Expression of phospholemman and its association with Na+-K+-ATPase in skeletal muscle: effects of aging and exercise training.

Phospholemman (PLM) is a recently identified accessory protein of the Na(+)-K(+)-ATPase (NKA), with a high level of expression in skeletal muscle. The objectives of this study are to characterize the PLM in skeletal muscle and to test the hypothesis that, as an accessory protein of NKA, expression of PLM and its association with the alpha-subunits of NKA is regulated during aging and with exercise training. PLM was characterized in skeletal muscle of 6- and 16-mo-old sedentary middle-aged rats (Ms), and the effects of aging and exercise training were studied in Ms, 29-mo-old sedentary senescent, and 29-mo-old treadmill-exercised senescent rats. Expression of PLM was muscle-type dependent, and immunofluorescence study showed that PLM distributed predominantly on the sarcolemmal membrane of the muscle fibers. Anti-PLM antibody reduced activity of NKA, and thus PLM appears to be required for NKA to express its full activity in skeletal muscle. Expression of PLM was not altered with aging but increased after exercise training. Coimmunoprecipitation studies demonstrated that PLM associates with both the alpha(1)- and alpha(2)-subunit isoforms of NKA. Compared with Ms rats, levels of PLM-associated alpha(1)-subunit increased in 29-mo-old sedentary senescent rats, and treadmill exercise has a tendency to partially reverse it. There was no significant change in PLM-associated alpha(2)-subunit with age, and exercise training has a tendency to increase that level. It is concluded that, in skeletal muscle, PLM appears to be a protein integral to the NKA complex and that PLM has the potential to modulate NKA in an isoform-specific and muscle type-dependent manner in aging and after exercise training.     

Diacylglycerol: Efficacy and Mechanism of Action of an Anti‐Obesity Agent

Obesity is at the forefront of global health issues and directly contributes to many chronic illnesses. Several dietary components show promise in the treatment of obesity, one of which is oil rich in diacylglycerols (DAGs). Present objectives are to examine scientific knowledge concerning DAG to assess evidence supporting the effects on substrate oxidation rates, body weight and fat mass, and blood lipids, and to assess safety, as well as elucidate potential mechanisms of action. DAG can be synthesized by an enzymatic process to produce mainly 1, 3‐isoform DAG. This 1, 3‐DAG oil is believed to have the ability to increase β‐oxidation, to enhance body weight loss, to suppress body fat accumulation, and to lower serum triacylglycerol levels postprandially. While certain animal and human studies indicate that consumption of 1, 3‐DAG has positive physiological effects, others report no effect. The mechanisms of action of DAG are suggested to decrease the resynthesis of chylomicrons as well as shunting them directly to the liver through the portal vein, where they are oxidized. This increased fat oxidation may influence control of food intake by increasing satiety. Further study into the precise mechanism is required to understand its effects. Safety studies show no risks in consuming a diet rich in DAG oil. Overall, consumption of oils with higher amounts of DAG, specifically 1, 3‐DAG, may be useful in the battle against obesity.     

Proteomic analysis of dental tissues

Teeth are highly refined structures formed by several types of specialised cell. Tooth formation embraces many areas of biomedical interest, including cellular mechanisms for calcium handling, protein secretion and mineralised tissue production. Proteomics offers great potential to elucidate these cellular roles, and to establish their relevance to general cell types. Here we review our proteomic investigations of dental enamel formation, covering both the approaches taken and some findings of general biomedical relevance.     

Synthesis of N,N',N"-trisubstituted thiourea derivatives and their antagonist effect on the vanilloid receptor

Twenty-seven N,N',N"-trisubstituted thiourea derivatives were prepared. Among them, 1-[3-(4'-hydroxy-3'-methoxy-phenyl)-propyl]-1,3-diphenethyl-thiourea (8l, IC(50)=0.32 microM), showed 2-fold higher antagonistic activity than that of capsazepine (3, IC(50)=0.65 microM) against the vanilloid receptor in a (45)Ca(2+)-influx assay.     

Changes induced by 6-hydroxydopamine in the paraventricular nucleus

Summary Remodelling of catecholaminergic (CA) fibers after cerebral intraventricular 6-hydroxydopamine (6-OH-DA) administration was evaluated quantitatively in the paraventricular nucleus (PAR) of young adult rats, using fluorescence microscopy (FM) and electron microscopy (EM). Fluorescent CA varicosities and CA boutons (marked with 5-OH-DA) were counted after survival periods of 4, 21, 56 or 180 days. Four days after 6-OH-DA treatment, the number of fluorescent varicosities dropped to 45% of control numbers but was restored to 79% of control values by 180 days. In the EM study, marked boutons had dropped more dramatically: to 12% of control numbers, after 4 days and 54% by 180 days post-neurotoxin. These data provide strong evidence that substantial but incomplete restoration of CA terminals occurred in PAR. It is of interest that, in all survival intervals, percentage reductions in numbers of CA terminals were more extreme when EM was used for quantification. Nevertheless, the trends indicating partial restoration of terminal numbers with time were parallel in the FM and EM studies. Structures identified as CA growth cones in PAR contained a feltwork of fine filaments together with mitochondria, granular vesicles (often with electron-dense cores marked by the 5-OH-DA label), vacuoles and smooth-surfaced reticulum. The presence of growth cones, some of which persisted 11 months after neurotoxin administration, further supports the inference that a regenerative response of CA elements was evoked in PAR by the 6-OH-DA treatment.Presented in part at IV International Catecholamine Symposium in California, September 1978     

Endurance training alters outward K+ current characteristics in rat cardiocytes.

The effect of endurance run training on outward K+ currents with rapidly inactivating (I(to)) and sustained or slowly inactivating (I(sus)) characteristics was examined in left ventricular (LV) cardiocytes isolated from sedentary (Sed) and treadmill-trained (Tr) female Sprague-Dawley rats. Isolated LV cardiocytes were used in whole cell patch-clamp studies to characterize whole cell I(to) and I(sus). Peak I(to) was greatest in cells isolated from the Tr group. When I(to) was corrected for cell capacitance to yield a current density, most, but not all, of the Sed vs. Tr differences in I(to) magnitude were eliminated. Regardless of how I(to) was expressed (e.g., I(to) or I(to) density), the time required to achieve a peak value was markedly shortened in the cardiocytes isolated from the Tr group. Training elicited a reduction in I(sus) density. Action potential characteristics were determined in Sed and Tr cardiocytes in primary culture. Training did not affect resting membrane potential, whereas peak membrane potential was reduced and time to peak membrane potential was prolonged in the Tr group. In addition, time to 50% repolarization was significantly increased in cells from the Tr group. Collectively, these data indicate that I(to) and I(sus) characteristics are altered by training in isolated LV cardiocytes. These alterations in I(to) and I(sus) may be responsible, at least in part, for the training-induced alterations in action potential configuration in cardiocytes in primary culture.     

An improved method for perfusion fixation of neural tissues for electron microscopy

A method employing vascular perfusion for improved preservation of biological ultrastructure is described, and its effectiveness demonstrated for mammalian nervous tissues. Following a physiological saline flush into the aorta, hydrogen peroxide and glutaraldehyde in phosphate buffer are perfused. After buffer rinses, tissue blocks are postfixed in osmic acid and potassium ferrocyanide. The success rate is enhanced greatly by close attention to details of perfusion technique. Advantages of the method include more uniform and complete preservation. In particular, superior images of membranous elements, glycogen granules and basal laminar material are achieved. Adjustments in osmolality may render the procedure suitable for non-mammalian forms and other tissues.     

Variations in atrioventricular valve innervation in four species of mammals

In this series of studies, the innervation patterns of whole-mount preparations of bicuspid and tricuspid valves were studied by light microscopy in the mouse, rat, guinea pig, and opossum. The acetylcholinesterase-positive networks of nerve fibers showed many similarities in the basic patterns of valve innervation in all of the species studied, but several interspecies variations were observed. The basal zone of the valve adjacent to the fibromuscular atrioventricular ring displayed the most dense plexus of nerves, with acetylcholinesterase-positive fibers being seen across the width of the valve. In the intermediate zone of the valve, less dense plexuses of nerve fibers were found; and these were more numerous in the cuspal areas and less numerous in the intervening commissural areas. In the distal portions of the valve, nerve networks arborized extensively, with some of their nerve fibers extending toward the chordae tendineae and the free edges of the valve cusps. Only in the guinea pig and opossum did these fibers reach the free margin of the valve cusp, where they either ended directly as free nerve endings or lay parallel to the free edge of the cusp, often running between adjacent chordae tendineae. Although the patterns of innervation were similar in both bicuspid and tricuspid valves, the innervation density of the bicuspid valve was greater than that of the tricuspid valve for each species examined. A distinguishing feature of guinea pig and opossum tricuspid valves was that their chordae tendineae were relatively more prominent and more densely innervated than the bicuspid chordae tendineae. Free nerve endings with no light microscopic evidence of specialization were present throughout the bicuspid and tricuspid valves of all species studied. Some nerve endings in the opossum showed evidence of specialization, with brush-like arborizations leading to presumed free terminals seen chiefly in the distal zone of the valve cusps. Although some general tendencies were apparent, we have demonstrated that interspecies heterogeneity exists in the terminal networks of the atrioventricular valves of mouse, rat, guinea pig, and opossum.     

Glibenclamide improves postischemic recovery of myocardial contractile function in trained and sedentary rats.

In this study, we sought to determine whether there was any evidence for the idea that cardiac ATP-sensitive K+ (K(ATP)) channels play a role in the training-induced increase in the resistance of the heart to ischemia-reperfusion (I/R) injury. To do so, the effects of training and an K(ATP) channel blocker, glibenclamide (Glib), on the recovery of left ventricular (LV) contractile function after 45 min of ischemia and 45 min of reperfusion were examined. Female Sprague-Dawley rats were sedentary (Sed; n = 18) or were trained (Tr; n = 17) for >20 wk by treadmill running, and the hearts from these animals used in a Langendorff-perfused isovolumic LV preparation to assess contractile function. A significant increase in the amount of 72-kDa class of heat shock protein was observed in hearts isolated from Tr rats. The I/R protocol elicited significant and substantial decrements in LV developed pressure (LVDP), minimum pressure (MP), rate of pressure development, and rate of pressure decline and elevations in myocardial Ca(2+) content in both Sed and Tr hearts. In addition, I/R elicited a significant increase in LV diastolic stiffness in Sed, but not Tr, hearts. When administered in the perfusate, Glib (1 microM) elicited a normalization of all indexes of LV contractile function and reductions in myocardial Ca(2+) content in both Sed and Tr hearts. Training increased the functional sensitivity of the heart to Glib because LVDP and MP values normalized more quickly with Glib treatment in the Tr than the Sed group. The increased sensitivity of Tr hearts to Glib is a novel finding that may implicate a role for cardiac K(ATP) channels in the training-induced protection of the heart from I/R injury.