Miguel Huerta

Astrocytic and microglia cells reactivity induced by neonatal administration of glutamate in cerebral cortex of the adult rats

Recent studies confirm that astrocytes and neurons are associated with the synaptic transmission, particularly with the regulation of glutamate (Glu) levels. Therefore, they have the capacity to modulate the Glu released from neurons into the extracellular space. It has also been demonstrated an intense astrocytic and microglia response to physical or chemical lesions of the central nervous system. However, the persistence of the response of the glial cells in adult brain had not been previously reported, after the excitotoxic damage caused by neonatal dosage of monosodium glutamate (MSG) to newborn rats. In this study, 4 mg/g body weight of MSG were administered to newborn rats at 1, 3, 5, and 7 days after birth, at the age of 60 days the astrocytes and the microglia cells were analyzed with immunohistochemical methods in the fronto‐parietal cortex. Double labeling to glial fibrillary acidic protein (GFAP) and BrdU, or isolectin‐B4 and BrdU identified astrocytes or microglia cells that proliferated; immunoblotting and immunoreactivity to vimentin served for assess immaturity of astrocytic intermediate filaments. The results show that the neonatal administration of MSG‐induced reactivity of astrocytes and microglia cells in the fronto‐parietal cortex, which was characterized by hyperplasia; an increased number of astrocytes and microglia cells that proliferated, hypertrophy; increased complexity of the cytoplasm extension of both glial cells and expression of RNAm to vimentin, with the presence of vimentin‐positive astrocytes. This glial response to neuroexcitotoxic stimulus of Glu on the immature brain, which persisted to adulthood, suggests that the neurotransmitter Glu could trigger neuro‐degenerative illnesses.

UVB photoprotection with antioxidants: Effects of oral therapy with d-α-tocopherol and ascorbic acid on the minimal erythema dose

Ultraviolet radiation absorption is responsible for the production of free radicals in damaged cells. This side effect may be neutralized using antioxidant substances. It has been reported that ascorbic acid and d-alpha-tocopherol scavenge reactive oxygen species. In a single-blind controlled clinical trial we studied 45 healthy volunteers divided into three groups. Group 1 received d-alpha-tocopherol 1,200 I.U. daily; Group 2 ascorbic acid 2 g daily and Group 3 ascorbic acid 2 g plus d-alpha-tocopherol 1,200 I.U. daily. Treatment was sustained for one week. Before and after treatment, the minimal erythema dose was determined in all participants. The results show that the median minimal erythema dose increased from 60 to 65 mJ/cm2 in Group 1 and from 50 to 70 mJ/cm2 in Group 3. No modifications were observed in Group 2. We conclude that d-alpha-tocopherol prescribed in combination with ascorbic acid produces the best photoprotective effect.

Effects of external calcium on potassium contractures in tonic muscle fibers of the frog (Rana pipiens).

K+ contractures of tonic bundles from cruralis muscle of the frog were studied with different K+ concentrations (10‐120 mM). K+ contractures had an initial transient phase followed by a sustained tension. The amplitude of the sustained tension diminished with high K+ concentration (80‐120 mM). However, in all cases, tension was maintained for several minutes. External Ca2+ reduction practically abolished the sustained phase of the K+ contractures. The initial phase was also reduced and tension spontaneously relaxed. The curve relating the peak tension with log [K+]o, showed that the threshold was not affected but the peak tension was reduced to about 70% in low‐Ca2+ saline (0 Ca2+ + 3 mM‐Mg2+) and 50% in Ca2+‐free saline (1 mM‐EGTA + 3 mM‐Mg2+). The dependence of the sustained tension on external Ca2+ was further confirmed by Ca2+ withdrawal and re‐establishment and/or by Ni2+ substitution for Ca2+ before or during K+ contractures. These results indicate that external Ca2+ had to be continuously present to maintain the tension during K+ contractures and that Ni2+ was not able to restore the normal temporal course of K+ contracture. The sustained phase was diminished by blocking agents of Ca2+ channels, such as nifedipine (1 microM) and diltiazem (1‐10 microM). The present results can be explained by a direct control of the Ca2+ currents on K+ contracture or by specific interactions between external Ca2+ and Ca2+‐binding sites in the membrane.

Selective reinnervation of twitch and tonic muscle fibres of the frog.

The electrical properties and innervation of piriformis muscle fibres and the conduction velocities and thresholds of the corresponding motor axons were studied. In normal muscles all fibres clearly fell into the category of twitch or tonic. Tonic muscle fibres were selectively reinnervated by small motor axons after crushing or cutting the piriformis nerve. Twitch fibres were reinnervated by large motor axons. Tonic fibres were also selectively reinnervated by small motor axons when the proximal stump of the piriformis nerve was cut and attached to the caudal end of the muscle. With this procedure the possibility of mechanical guidance by remaining neural sheaths was eliminated. Polyneuronal innervation in twitch fibres in piriformis muscles of normal frogs was 4.7% and in contralateral muscles of operated frogs with the piriformis nerve cut it was 22.6%. Unexpectedly three out of seventeen tonic fibres in contralateral muscles were simultaneously innervated by both small and large motor axons. It is concluded that reinnervation is highly specific for fibre type in frog skeletal muscles.

Skeletal muscle Ca2+ channels

SummaryCa2+ channels are widely distributed among different cell types. We shall describe in this paper kinetic properties of voltage-dependent slow Ca2+ channels in mammalian and frog skeletal muscle fibres. In addition, recent data on a fast-activated Ca2+ channel will be presented. Finally, the possible physiological role of the channel will be considered.

Potassium and caffeine contractures in fast and slow muscles of the chicken.

1 K+ contractures, caffeine contractures and electrical properties were studied in slow (posterior latissimus dorsi; p.l.d.) and fast (anterior latissimus dorsi; a.l.d.) chicken muscles. 2. P.l.d. K+ contractures show a transient increase of tension that relaxes spontaneously. Contractures in a.l.d. show an initial component followed by a maintained tension. 3. A.l.d. K+ contractures of similar amplitude and time course were reproduced at 4 min intervals. In p.l.d., the interval needed for full recovery is about 30 min. In Cl‐free saline p.l.d. and a.l.d. K+ contractures can be reproduced at 4 min intervals. 4. The time course of repolarization after a short exposure to 160 mM‐KCl was much slower in p.l.d. than in a.l.d. In Cl‐free saline the time course of repolarization becomes faster in p.l.d. 5. The membrane resistance was not modified in a.l.d. and was increased in p.l.d. by Cl‐free saline. The calculated Cl‐ conductance in p.l.d. was about 70% of the total membrane conductance. 6.In a.l.d., Mn2+, D600 and external Ca2+ reduction greatly diminishes the maintained phase of the K+ contracture leaving the initial phase almost unmodified. Under similar conditions p.l.d. K+ contractures were slightly reduced. 7. P.l.d. caffeine contractures (10‐40 mM) were not maintained and they were not modified by Ca‐free saline, Cd2+, Co2+, Mn2+ and D600. 8. A.l.d. caffeine contractures (2‐15 mM) were maintained and were highly dependent on external Ca2+. In addition they were greatly reduced by Cd2+, Co2+, Mn2 and D600. 9. It is suggested that caffeine contractures of a.l.d. are elicited by a Ca2+ entry into the muscle from the external fluid.

Calcium action potentials and calcium currents in tonic muscle fibres of the frog (Rana pipiens).

Slow action potentials were evoked in cruralis tonic and twitch fibres of the frog after drastically reducing the Cl‐ and K+ conductances. Tonic fibres were identified by their electrical characteristics. They had an effective resistance (Reff) of 50 +/‐ 6 M omega (n = 27) and a membrane time constant (tau m) of 440 +/‐ 70 ms (n = 8). In twitch fibres Reff = 2.9 +/‐ 0.3 M omega (n = 16) and tau m = 50 +/‐ 4 ms (n = 6). In tonic fibres the slow action potential had a threshold of ‐50 to ‐60 mV and a peak amplitude of ‐10 mV. In twitch fibres the slow action potential had a threshold of ‐40 mV and reached a peak amplitude of +40 mV. The responses were blocked by the addition of Cd2+ (2 mM) or Co2+ (5 mM). These results strongly suggest that Ca2+ is the main carrier of current during the response. Using the three‐micro‐electrode voltage‐clamp technique a slow inward membrane current underlying the Ca2+ potential could be described in tonic muscle fibres. The slow inward current was mainly carried by Ca2+, since it was reduced when external Ca2+ concentration was lowered or when Cd2+ (2 mM) was added. Moreover, Ca2+ was the only cation in the solution that could carry inward current. It had a mean threshold of ‐60 mV, reached a maximum value at ca. 0 mV, ranged from 24 to 28 microA/cm2 and had a mean reversal potential of +35 mV. In about half of the examined tonic fibres inward current declined with time, only slowly. This can either be explained by there being less contamination by K+ outward current, or by the presence of two types of Ca2+ channels in the tonic fibre membrane.

Effect of chronic administration of forskolin on glycemia and oxidative stress in rats with and without experimental diabetes.

Forskolin is a diterpene derived from the plant Coleus forskohlii. Forskolin activates adenylate cyclase, which increases intracellular cAMP levels. The antioxidant and antiinflammatory action of forskolin is due to inhibition of macrophage activation with a subsequent reduction in thromboxane B2 and superoxide levels. These characteristics have made forskolin an effective medication for heart disease, hypertension, diabetes, and asthma. Here, we evaluated the effects of chronic forskolin administration on blood glucose and oxidative stress in 19 male Wistar rats with streptozotocin-induced diabetes compared to 8 healthy male Wistar rats. Rats were treated with forskolin, delivered daily for 8 weeks. Glucose was assessed by measuring fasting blood glucose in diabetic rats and with an oral glucose tolerance test (OGTT) in healthy rats. Oxidative stress was assessed by measuring 8-hydroxydeoxyguanosine (8‑OHdG) in 24-h urine samples. In diabetic rats, without forskolin, fasting blood glucose was significantly higher at the end than at the beginning of the experiment (8 weeks). In both healthy and diabetic rats, forskolin treatment lowered the fasting glucose at the end of the experiment but no effect was found on oral glucose tolerance. The 8-OHdG levels tended to be less elevated in forskolin-treated than in untreated group. Our results showed that chronic administration of forskolin decreased fasting blood glucose levels; however, the reductions of 8-OHdG were not statistically significant.

Effects of Cannabinoids on Synaptic Transmission in the Frog Neuromuscular Junction

This study aimed to investigate the function of the cannabinoid receptor in the neuromuscular junction of the frog (Rana pipiens). Miniature end-plate potentials were recorded using the intracellular electrode recording technique in the cutaneous pectoris muscle in the presence of the cannabinoid agonists WIN55212-2 (WIN; R-(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)]-pyrolol[1,2,3de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone) and arachidonylcyclopropylamide [ACPA; N-(2-cyclopropyl)-5Z,8Z,11Z,147-eicosatetraenamide] and the cannabinoid antagonists 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide (AM281) and 6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)methanone (AM630). Adding WIN to the external medium decreased the frequency and amplitude of the miniature end-plate potentials (MEPPs); the WIN EC50 value was 5.8 ± 1.0 μM. Application of ACPA, a selective agonist of cannabinoid receptor CB1, also decreased the frequency of the MEPPs; the ACPA EC50 value was 115.5 ± 6.5 nM. The CB2 antagonist AM630 did not inhibit the effects of WIN, indicating that its action is not mediated through the CB2 receptor. However, the CB1 antagonist AM281 inhibited the effects of WIN and ACPA, suggesting that their actions are mediated through the CB1 receptor. Pretreatment with the pertussis toxin inhibited the effects of WIN and ACPA, suggesting that their effects are mediated through Gi/o protein activation. The N-type Ca2+ channel blocker ω-conotoxin GVIA (ω-CgTX) diminished the frequency of the MEPPs, with an ω-CgTX EC50 value of 2.5 ± 0.40 μM. Blocking the N-type Ca2+ channels with 5 μM ω-CgTX before addition of ACPA to the bath had no additional inhibitory effect on the MEPPs, whereas in the presence of 1 μM ω-CgTX, ACPA had an additional inhibition effect. These results suggest that cannabinoids modulate transmitter release in the end-plate of the frog neuromuscular junction by activating CB1 cannabinoid receptors in the nerve ending.

Cannabinoid receptor type 1 activation by arachidonylcyclopropylamide in rat aortic rings causes vasorelaxation involving calcium-activated potassium channel subunit alpha-1 and calcium channel, voltage-dependent, L type, alpha 1C subunit

Cannabinoids are key regulators of vascular tone, some of the mechanisms involved include the activation of cannabinoid receptor types 1 and 2 (CB); the transient receptor potential cation channel, subfamily V, member 1 (TRPV1); and non-(CB(1))/non-CB2 receptors. Here, we used the potent, selective CB(1) agonist arachidonylcyclopropylamide (ACPA) to elucidate the mechanism underlying vascular tone regulation. Immunohistochemistry and confocal microscopy revealed that CB(1) was expressed in smooth muscle and endothelial cells in rat aorta. We performed isometric tension recordings in aortic rings that had been pre-contracted with phenylephrine. In these conditions, ACPA caused vasorelaxation in an endothelium-independent manner. To confirm that the effect of ACPA was mediated by CB(1) receptor, we repeated the experiment after blocking these receptors with a selective antagonist, AM281. In these conditions, ACPA did not cause vasorelaxation. We explored the role of K(+) channels in the effect of ACPA by applying high-K(+) solution to induce contraction in aortic rings. In these conditions, the ACPA-induced vasorelaxation was about half that observed with phenylephrine-induced contraction. Thus, K(+) channels were involved in the ACPA effect. Furthermore, the vasorelaxation effect was similarly reduced when we specifically blocked calcium-activated potassium channel subunit alpha-1 (KCa1.1) (MaxiK; BKCa) prior to adding ACPA. Finally, ACPA-induced vasorelaxation was also diminished when we specifically blocked the calcium channel, voltage-dependent, L type, alpha 1C subunit (Ca(v)1.2). These results showed that ACPA activation of CB(1) in smooth muscle caused vasorelaxation of aortic rings through a mechanism involving the activation of K(Ca)1.1 and the inhibition of Ca(v)1.2.