Decrease of rotenone inhibition is a sensitive parameter of complex I damage in brain non-synaptic mitochondria of aged rats.

We investigated NADH oxidation in non-synaptic and synaptic mitochondria from brain cortex of 4- and 24-month-old rats. The NADH oxidase activity was significantly lower in non-synaptic mitochondria from aged rats; we also found a significant decrease of sensitivity of NADH oxidation to the specific Complex I inhibitor, rotenone. Since the rotenone-binding site encompasses Complex I subunits encoded by mtDNA, these results are in accordance with the mitochondrial theory of aging, whereby somatic mtDNA mutations are at the basis of cellular senescence. Accordingly, a 5 kb deletion was detected only in the cortex of the aged animals.

Beta-adrenoceptor changes in submandibular glands of old mice.

The hypothesis that modifications in beta-adrenergic receptors may be responsible for age-dependent change previously observed in vivo, has been investigated. Beta-adrenoceptor characteristics of submandibular glands of mice were studied by using the beta-adrenergic antagonist (-)-[3H]dihydroalprenolol. Data from such studies indicated the presence of two functional populations of binding sites in membrane preparations from young animals, displaying high and low affinity, respectively. Experiments performed on old mice membrane preparations revealed a 50% decrease in the high-affinity population receptor number when compared to the preparations from young animals. However, the affinity did not change significantly with advancing age. With regard to the low-affinity population, no statistically significant changes were observed. From these data it can be reasonably assumed that beta-adrenoceptor alteration during ageing may play a major role in the age-dependent impairment of beta-adrenergic responses in vivo.

Age-related alterations of isoproterenol-stimulated adenylyl-cyclase activity are partially corrected by thymic graft.

Previous experimental results have demonstrated progressive impairments in beta-adrenergic responsiveness with advancing age. Beta-adrenoceptors are involved in the alterations as their density progressively decreases during aging. Alterations in both in vivo responsiveness and receptor density are corrected by neonatal thymic grafts. In the present paper adenylyl-cyclase (AC) activity has been studied in the same animal models used before. Results show that no statistically significant changes can be observed when AC is assayed in absence of beta-adrenergic stimulation. On the contrary, when assayed after Isoproterenol stimulation, AC activity shows a shift of the peak and a decrease of its height in aged animals. A neonatal thymus grafted into old recipients one month before the experiment was performed, is capable of correcting the altered height of the peak but not the peak concentration.

Immune enhancement by conditioning of senescent mice. Comparison of old and young mice in learning ability and in ability to increase natural killer cell activity and other host defense reactions in response to a conditioned stimulus.

It has been clearly demonstrated that immune responses may be conditioned in a manner similar to that of the classical Pavlovian experiments. Evidence of impaired immune function in aging has raised the question of whether psychological conditioning of an immune response can also be effective in old age. The knowledge that aged mice have decreased spleen cell natural killer (NK) activity and that NK cytotoxicity, at least in young mice, can be psychologically conditioned led us to explore in old mice the possibility of conditioning the response of NK cell activity using the odor of camphor as the conditioned stimulus (CS) and the injection of Poly I:C as the unconditioned stimulus (US). Young and old male mice were divided into five and six groups, respectively. They received the CS and/or the US in association (conditioning) trials (sessions 1-9). Mice were exposed to the camphor odor alone at 72 hours after the final association trial to observe the conditioning phenomenon (session 10). The group conditioned with Poly I:C and camphor and receiving the CS at session 10 showed statistically significant increases in spleen cell NK activity over those of the control groups that did not receive the CS treatment at session 10 (2.6- and 4.0-fold increase in young and old, respectively). Treatment with camphor odor alone had no effect on boosting NK cell activity. These findings demonstrate the possibility of conditioning immune responses in old age, offering a valuable tool for attenuating age-related immune deterioration in various species, including the human. In addition, these results again confirm highly significant immune enhancement by classical conditioning and extend previous findings from female mice to males as well.

Influence of aging and thymus on the beta-adrenergic dependent adenylyl cyclase activity in mouse brain cortex.

Beta-adrenoceptor (betaAR) density has previously been found altered in brain cortex of aging mice. In the present paper the question has been addressed whether adenylyl cyclase (AC) also presents similar age-related changes. Due to the fact that age-related receptor impairments have previously been corrected by thymic grafts, the influence of thymus was also studied. Therefore, experiments were performed on young, athymic nude young, old and thymus-grafted old mice. BetaAR characteristics and basal and isoproterenol-stimulated AC activity were assayed in the same membrane preparations. Our results confirm a decrease of receptor density of beta(1) subtype in both old and athymic nude young mice, paralleled by a decreased isoproterenol-stimulated AC activity. A neonatal thymus grafted into old recipients is able to correct the changes in receptor density and the height of the peak of AC response. The location of the peak, however, remains shifted to the right, as it occurs in nude and in untreated old mice. It can be concluded that alterations may occur at different steps along the stimulus transducing chain, as suggested by the differential effect induced by thymus on receptor changes, which are completely recovered, and AC activity alterations, only partially corrected.

Parallel decrease of adenylyl cyclase activity and beta1-adrenoceptor density in brain cortex of aging mice.

Beta-adrenergic receptors (betaARs) and adenylyl cyclase (AC) activity have been found to undergo progressive alterations in the brain cortex of aging mice. In particular, betaAR changes are in charge of the beta1 subpopulation (beta1AR), beta2-adrenoceptors (beta2ARs) showing no age-related impairments. On these bases, the question arises whether AC alterations can be accounted for by beta1AR changes or, alternatively, whether there are also receptor independent AC modifications. Experiments have thus been performed on the brain cortex from young and old mice assaying beta1AR and beta2AR characteristics and isoproterenol (IPR) and forskolin stimulated AC activity in the same membrane preparations. Other membranes were previously treated with the reducing agent dithiothreitol (DTT) which is known to specifically destroy the binding capacity of beta1ARs. No statistically significant differences in basal AC activity have been found among the groups studied. Data on IPR-stimulated AC activity in old animals confirm previous findings demonstrating impaired responsiveness when compared with that of young mice. DTT treated membrane preparations from both young and old mice show decreased AC activities. The entity of the decrease is lower in aged animals due to the lower initial level of beta1ARs. Forskolin stimulation, which is assumed to directly activate AC, has been found impaired in aged mice when compared with young animals, suggesting that also post-receptor alterations occur with advancing age.

Ageing and thymus-induced differential regulation of beta 1 and beta 2 adrenoceptors of mouse brain cortex.

Two questions have been addressed by the present paper, that is, the differential influence of age on beta 1 and beta 2 receptors and the thymus-induced recovery of such changes in receptor density and affinity have been assayed in brain cortex of Balb/c-nu mice of different ages. Results have shown a progressive decrease of beta 1 receptor density with advancing age, while no statistically significant changes were observed in beta 2 receptor density. Receptor affinity did not show any changes among the various groups examined. The influence of the thymus on receptor characteristics has been studied comparing young, old and thymus-grafted old mice. Total receptor density, which is decreased in old animals, can be up-regulated by thymus graft in old recipients. Interestingly, such a corrective effect is exerted only on the beta 1 population, the beta 2 receptor not being significantly affected. Thymic graft, therefore, acts just on the population which is found altered during ageing.

Age-dependent decrease of beta-adrenoceptor density in the submandibular glands of mice and its modulation by the thymus.

Beta-adrenergic receptors were characterized in submandibular glands of ageing mice and old mice grafted with a neonatal thymus. No statistically significant changes of receptor affinity were found in the animal models investigated. On the contrary, receptor density showed a progressive decrease with advancing age. The age-related decrease has been found partially corrected in thymus-grafted old animals, which show a statistically significant recovery of receptor density when compared to their untreated littermates. Receptor modulation can be responsible for the age-related impairment and the thymus-dependent correction of beta-adrenergic responsiveness of submandibular glands previously observed in vivo. Hormonal balance and thyroid hormones, in particular, are suggested as being involved in the age- and thymus-dependent regulation of receptor density. In the accompanying Appendix, we describe the mathematical method used to calculate both specific and nonspecific binding from total binding data.

Centrophenoxine-induced increase of beta-adrenoceptor density in brain cortex of old mice.

The influence of centrophenoxine treatment on beta-adrenoceptor density has been studied in the brain cortex of old mice. One group of 21-mth-old mice was injected intraperitoneally with 3.8 mg/day centrophenoxine daily for 5 days. A second group received the same daily treatment for 40 days. Two other groups of untreated mice of the same age served as controls. Receptor affinity showed no statistically significant changes in the four different groups investigated. As regards the receptor density, an increasing trend has been elicited between the 5-day treated mice and their controls; however, the increase was not statistically significant. The second group of old mice treated with centrophenoxine for 40 days exhibited a statistically significant increase in receptor density when compared with untreated animals. The modulation of beta-adrenoceptor density may be explained as the result of either a direct action on synaptic structure or an indirect effect mediated by thyroid hormones.

Thyroid hormone-induced up-regulation of beta1- and beta2-adrenergic receptors during aging.

Strong experimental evidence suggests that thyroid hormones influence beta-adrenoceptor regulation. The question arises whether in tissues like mouse brain cortex bearing both beta1- and beta2-subpopulations, these receptor subtypes may be regulated independently, as demonstrated after some drug stimulations. An eventual influence of aging on triiodothyronine (T(3))-induced subpopulation response is also worth studying, because of the decrease of receptor density in old animals observed only in the beta1-type. These points have been addressed in the present paper, performing experiments on young and old mice stimulated with a single injection of T(3). The response has been assayed as rapidly as 15 min after T(3) treatment. Results on untreated control mice confirm previous findings showing that the decrease of receptor density is in charge only of the beta1-subtype. T(3) stimulation induces up-regulation of both beta1- and beta2-adrenoceptors in young animals. The response shows no impairment with aging, as in both receptor subtypes a significant increase can be demonstrate. Due to the very short time of response, and unmasking mechanism can be hypothesized for receptor up-regulation, though an increase of protein synthesis cannot be excluded.

Thymic regulation of brain cortex beta-adrenoceptors during development and aging.

The influence of the thymus on beta-adrenoceptors has been studied in the brain cortex of mice during developing and aging. Affinity of beta-adrenoceptors shows no statistically significant changes in the various animal models investigated. Receptor density shows a fall in both athymic nude mice and in old normal mice. Receptor density, in particular, decreases progressively with advancing age. It has been demonstrated that thymus exerts a regulatory role in both development and aging, as a neonatal thymic graft is capable of reversing the receptor impairments found in young athymic nude mice and in old normal mice.

Impaired adaptive receptor regulation: an index of aging?

Many neuroendocrine functions are altered in old animals and their study may represent important steps in the understanding of the mechanisms of aging. A deeper insight, however, can be achieved by investigating the responsiveness to stimuli, which may reveal alterations not evident in the unstimulated conditions. At this level of study, many of such impairments have been found to be caused by receptor changes. In the present paper a third level of study is suggested in order to gain evidence of some remote failure of adaptive processes strictly linked to intimate mechanisms of aging. As at the second level of study different receptor characteristics can frequently be found at the basis of age-related alterations of biological responsiveness, at the proposed third level altered capacity of receptor regulation may be hypothesized as responsible for altered cell adaptation following hormone and drug stimuli. Experimental data are given which support this view. The possibility that receptor regulation may be used as an index of aging is suggested. This hypothesis leads to the problem of judging the validity of biological parameters deputed to represent good indices of aging. In order to solve this problem, the potential use of a mathematical model of mortality kinetics is discussed.

The human lymphocyte as a model of beta-adrenoceptor regulation in aging and disease.

Extensive experimental evidence suggests that human lymphocytes may model the behavior of other tissues as far as beta-adrenoceptor characteristics are concerned. In fact, although lymphocytes bear only beta2-adrenoceptor subtype, they may mirror other tissues in various physiological and pathological conditions. In the present paper examples are given demonstrating receptor regulations after drug or surgical stimulus. Age-related changes have also been taken into account. Treatment with beta1-selective beta-blockers without or with intrinsic sympathomimetic activity induces, respectively, up- or down-regulation of beta-adrenoceptors of lymphocytes in agreement with other tissues, as suggested by clinical findings. Data on the influence of thyroidectomy are also in agreement with clinical observations and animal experimental findings on other tissues. Preliminary results on old subjects also suggest a possible role of the human lymphocyte in modelling other tissues or organs in relation to aging processes. The clinical application could be of paramount importance due to the impossibility to directly monitor the receptor status in most of human tissues.

Functional interrelationships in aging processes: alterations and reversibility.

The problem of assessing the relevance of the reversibility of age-related functional alterations for aging studies has been presented. Transduction mechanisms of adrenergic stimulation have been chosen as the target of age-related changes and thymus as the effector of some corrective interventions performed at advanced age. Both alterations of adrenoceptor characteristics and their reversibility have been reviewed. beta-adrenoceptors have been studied in organs bearing only one subtype of receptors or both, revealing an age-related decrease in density only in the beta1-subtype. It has been shown that a similar age-related decrease is present in alpha1-adrenoceptor density. Such alterations are corrected by grafting a neonatal thymus into old mice. On the contrary, thymus fails to correct the alteration of T4-induced upregulation of beta-adrenoceptors indicating some limits to its corrective effect when the net of functional interrelationships becomes relatively complex. Both failures and successes of thymic grafts and thymic extracts in reversing age-related changes are discussed taking into account the effects induced on the life span of the animals. Different unsolved problems stemming from the previous considerations are also presented. Among them the controversial question about linearity and non-linearity of biological parameters presumed to be good indices of aging is discussed, with the aid of a simple model as a schematic example.

Age-related regulation of mouse brain cortex adrenoceptors following camphor vapor exposition.

The paper deals with the ability of adrenergic receptors (AR) of mouse brain cortex to be differentially regulated in response to single or multiple expositions to camphor vapor. The regulation of alpha- and beta-adrenoceptors has been studied in young and old Balb/c-nu mice. Results confirm the decrease of total beta-adrenoceptor density previously observed in untreated mice with advancing age; in addition, receptor density decreases in both young and old mice after a single exposition to camphor vapor, followed by an adaptation after multiple stimuli. The beta1, subtype is mainly responsible for density decrease in young animals, while both beta1, and beta2 subtypes contribute to the decrease in old mice. On the contrary, beta2 subpopulation gives the major contribution to the adaptive recovery in both young and old mice. alpha-Adrenoceptors also show an age-related decrease in the control group; after a single exposition they show an increased density with the exception of alpha1-subset in the young group. Repeated expositions lead to a rather general adaptive response towards pre-stimuli conditions. The differential behaviour of receptor subtypes in response to camphor vapor exposition can be related to the differential alterations of receptor characteristics observed during aging and also suggests a possible mechanism through which these alterations may occur.

Exploring the damage limitation possibilities of mineral fibres for future integrated solutions: an in vitro study.

Owing to their possible carcinogenic effect, asbestos and other silica derivatives have been identified as priority substances for risk reduction and prevention of pollution. Neutralisation procedures have thus become a topical research subject in many European and American countries. In the present study, silica derivatives (asbestos-containing and asbestos substitutes like slag wool, rock wool, cement asbestos) were fully impregnated with an epoxy resin according to the procedure used for the in situ impregnation with viscous polymeric media, which penetrate and cement the fibres in place and reduce the risk of their dispersion in air. Untreated and treated samples were used to investigate their in vitro interaction with a human continuous epithelial cell line (NCTC 2544 keratinocytes) and test the resin's efficiency in passivating the surface activity of the fibrous particulate. SEM and morpho-quantitative data evidenced that impregnation with the epoxy resin modifies the mineral fibres' bioactivity (reduction of cell adhesion and decreased spread/round cell ratio) and demonstrated the value of in vitro cell testing after passivation as a risk-assessment procedure. These tests could be used for the rapid determination of the level of passivation of new synthetic mineral fibrous materials subjected to resin impregnation.