Assessment of airway hyperresponsiveness in murine tracheal rings.

Isolated tracheal rings have often been used to directly measure the contractile output of airway smooth muscle (ASM). Here, we describe the method for excising murine tracheas, mounting tracheal rings in organ baths, and measuring the isometric forces generated by the ASM when stimulated by drug additions or electric field stimulation. The apparatus for the setup and the pathways responsible for stimulation are detailed. Examples of the responses and analyses of two types of ASM stimulation are illustrated: (1) the carbachol concentration-response curve and (2) the frequency-response curve elicited by electric field stimulation.

In vitro measurements of tracheal constriction using mice.

Transgenic and knockout mice have been powerful tools for the investigation of the physiology and pathophysiology of airways(1,2). In vitro tensometry of isolated tracheal preparations has proven to be a useful assay of airway smooth muscle (ASM) contractile response in genetically modified mice. These in vitro tracheal preparations are relatively simple, provide a robust response, and retain both functional cholinergic nerve endings and muscle responses, even after long incubations. Tracheal tensometry also provides a functional assay to study a variety of second messenger signaling pathways that affect contraction of smooth muscle. Contraction in trachea is primarily mediated by parasympathetic, cholinergic nerves that release acetylcholine onto ASM (Figure 1). The major ASM acetylcholine receptors are muscarinic M2 and M3 which are G(i/o ;)and Gq coupled receptors, respectively(3,4,5). M3 receptors evoke contraction by coupling to Gq to activate phospholipase C, increase IP3 production and IP3-mediated calcium release from the sarcoplasmic reticulum(3,6,7). M2/G(i/o ;)signaling is believed to enhance contractions by inhibition of adenylate cyclase leading to a decrease in cAMP levels(5,8,9,10). These pathways constitute the so called "pharmaco-contraction coupling" of airway smooth muscle(11). In addition, cholinergic signaling through M2 receptors (and modulated by M3 signaling) involves pathways that depolarize the ASM which in turn activate L-type, voltage-dependent calcium channels (Figure 1) and calcium influx (so called "excitation-contraction coupling")(4,7). More detailed reviews on signaling pathways controlling airway constriction can be found(4,12). The above pathways appear to be conserved between mice and other species. However, mouse tracheas differ from other species in some signaling pathways. Most prominent is their lack of contractile response to histamine and adenosine(13,14), both well-known ASM modulators in humans and other species(5,15). Here we present protocols for the isolation of murine tracheal rings and the in vitro measurement of their contractile output. Included are descriptions of the equipment configuration, trachea ring isolation and contractile measurements. Examples are given for evoking contractions indirectly using high potassium stimulation of nerves and directly by depolarization of ASM muscle to activate voltage-dependent calcium influx (1. high K(+), Figure 1). In addition, methods are presented for stimulations of nerves alone using electric field stimulation (2. EFS, Figure 1), or for direct stimulation of ASM muscle using exogenous neurotransmitter applied to the bath (3. exogenous ACH, Figure 1). This flexibility and ease of preparation renders the isolated trachea ring model a robust and functional assay for a number of signaling cascades involved in airway smooth muscle contraction.

BK channel ß1 subunits regulate airway contraction secondary to M2 muscarinic acetylcholine receptor mediated depolarization.

The large conductance calcium- and voltage-activated potassium channel (BK channel) and its smooth muscle-specific ß1 subunit regulate excitation­contraction coupling in many types of smooth muscle cells. However, the relative contribution of BK channels to control of M2- or M3-muscarinic acetylcholine receptor mediated airway smooth muscle contraction is poorly understood. Previously, we showed that knockout of the BK channel ß1 subunit enhances cholinergic-evoked trachea contractions. Here, we demonstrate that the enhanced contraction of the BK ß1 knockout can be ascribed to a defect in BK channel opposition of M2 receptor-mediated contractions. Indeed, the enhanced contraction of ß1 knockout is eliminated by specific M2 receptor antagonism. The role of BK ß1 to oppose M2 signalling is evidenced by a greater than fourfold increase in the contribution of L-type voltage-dependent calcium channels to contraction that otherwise does not occur with M2 antagonist or with ß1 containing BK channels. The mechanism through which BK channels oppose M2-mediated recruitment of calcium channels is through a negative shift in resting voltage that offsets, rather than directly opposes, M2-mediated depolarization. The negative shift in resting voltage is reduced to similar extents by BK ß1 knockout or by paxilline block of BK channels. Normalization of ß1 knockout baseline voltage with low external potassium eliminated the enhanced M2-receptor mediated contraction. In summary, these findings indicate that an important function of BK/ß1 channels is to oppose cholinergic M2 receptor-mediated depolarization and activation of calcium channels by restricting excitation­contraction coupling to more negative voltage ranges.

BK channel beta1-subunit regulation of calcium handling and constriction in tracheal smooth muscle.

The large-conductance, Ca2+-activated K+ (BK) channels are regulators of voltage-dependent Ca2+ entry in many cell types. The BK channel accessory beta1-subunit promotes channel activation in smooth muscle and is required for proper tone in the vasculature and bladder. However, although BK channels have also been implicated in airway smooth muscle function, their regulation by the beta1-subunit has not been investigated. Utilizing the gene-targeted mice for the beta1-subunit gene, we have investigated the role of the beta1-subunit in tracheal smooth muscle. In mice with the beta1-subunit-knockout allele, BK channel activity was significantly reduced in excised tracheal smooth muscle patches and spontaneous BK currents were reduced in whole tracheal smooth muscle cells. Knockout of the beta1-subunit resulted in an increase in resting Ca2+ levels and an increase in the sustained component of Ca2+ influx after cholinergic signaling. Tracheal constriction studies demonstrate that the level of constriction is the same with knockout of the beta1-subunit and BK channel block with paxillin, indicating that BK channels contribute little to airway relaxation in the absence of the beta1-subunit. Utilizing nifedipine, we found that the increased constriction caused by knockout of the beta1-subunit could be accounted for by an increased recruitment of L-type voltage-dependent Ca2+ channels. These results indicate that the beta1-subunit is required in airway smooth muscle for control of voltage-dependent Ca2+ influx during rest and after cholinergic signaling in BK channels.

The influence of long-term Aloe vera ingestion on age-related disease in male Fischer 344 rats.

The effects of long-term Aloe vera ingestion on age-related diseases were investigated using male specific pathogen-free (SPF) Fischer 344 rats. Experimental animals were divided into four groups: Group A, the control rats fed a semi-synthetic diet without Aloe vera; Group B, rats fed a diet containing 1% freeze-dried Aloe vera filet; Group C, rats fed a diet containing 1% charcoal-processed, freeze-dried Aloe vera filet; and Group D, rats fed the control diet and given whole leaf charcoal-processed Aloe vera (0.02%) in the drinking water. This study demonstrates that life-long Aloe vera ingestion produced neither harmful effects nor deleterious changes. In addition, Aloe vera ingestion appeared to be associated with some beneficial effects on age-related diseases. Groups B exhibited significantly less occurrence of multiple causes of death, and a slightly lower incidence of fatal chronic nephropathy compared with Group A rats. Groups B and C rats showed the trend, slightly lower incidences of thrombosis in the cardiac atrium than Group A rats. Therefore, these findings suggest that life-long Aloe vera ingestion does not cause any obvious harmful and deleterious side effects, and could also be beneficial for the prevention of age-related pathology.

Assessing effects of long-term food restriction on myocardial energetics in the isolated heart preparation.

Food restriction (FR) may increase longevity by increasing the efficiency of energy utilization by some organs. We tested whether any effect of FR on the energy efficiency of isolated, isovolumically beating hearts could be observed, by studying four groups of rats: (1). AL fed (AL) 10-13-month-old rats, (2). age matched, FR at 60% of AL rats, (3). young AL, heart weight matched to FR rats and (4). 10-13-month-old AL rats, short-term FR for the last 3 weeks of life. The oxygen cost of tension development was not different among the groups. With contractility changed by calcium, the oxygen cost of contractility was higher in the young AL than in the adult rats either AL or short-term FR. With isoproterenol, it was higher in FR than in AL groups. The basal metabolic rate of hearts was higher in the adult AL than in the short-term, but not long-term, FR rats. In the long run, FR did not significantly change the pattern of cardiac energy utilization of isolated, isovolumically beating hearts. Our observations do not lend support to the hypothesis that the anti-aging action of FR is mediated by changes in cardiac efficiency.