Effective Drug Supply for Adaptive Clinical Trials: Recommendations by the DIA Adaptive Design Scientific Working Group Drug Supply Subteam.

During the past decade, there has been increasing interest in adaptive clinical trials in pharmaceutical drug development as a means to improved decision making, better dose selection, and reduction in cost and time to market. Nevertheless, the operational challenge of drug supply continues to be a barrier preventing greater uptake of adaptive designs. Such studies require the ability to quickly accommodate changes in treatment allocation while maintaining the integrity of the blind. The DIA Adaptive Design Scientific Working Group formed a subteam to discuss solutions to the drug supply dilemma. In this paper, the subteam discusses effective strategies for drug manufacturing, labeling, packaging, and randomization and addresses financial concerns.

Bronchial, alveolar, and vascular-induced anaphylaxis and irritant-induced cardiovascular and pulmonary responses.

We examine the respiratory, bronchomotor, cardiac, and vascular responses to histamine and ragweed allergen delivered to the bronchi or alveoli compartments and the potential role of sensory nerves and reflexes mediating the histamine-induced responses. The masses of aerosols deposited in the bronchi and alveoli were quantitated using radioaerosol techniques. Activation of sensory nerves and/or histamine-induced mediator release were characterized by depositing nedocromil sodium aerosol prior to histamine challenge. The histamine-induced responses due to vagosympathetic transmission were determined by performing bilateral vagotomy. Both histamine and ragweed increased respiratory rate, ventilation, and bronchomotor tone whether deposited in the bronchial or alveolar regions. However, these responses were not elicited when histamine was administered intravenously. Precipitous allergen-induced decreases in heart rate and systolic and diastolic pressure were maximal 72 sec following ragweed deposition in alveolar regions of the lungs. Increases in respiratory rate were mediated via the vagus whether delivered to the bronchi, alveoli, or vasculature. Histamine-induced increases in respiratory rate and bronchomotor tone were attenuated by nedocromil. When histamine was delivered to the alveolar regions, increases in lung resistance appeared to be mediated primarily via the vagus and when delivered to the bronchial airways primarily by its action on smooth muscle or local reflexes. Histamine-induced hypotension and bradycardia appear to be mediated by the direct action of histamine on the cardiovascular system rather than through a vagally mediated reflex.

The use of digital artery sympathectomy as a salvage procedure for severe ischemia of Raynaud's disease and phenomenon.

We retrospectively reviewed the effectiveness of digital artery sympathectomy as a last resort to prevent amputation in severe cases of Raynaud's disease and phenomenon. Seven patients underwent digital artery sympathectomy as a salvage procedure to prevent amputation. The patients developed cold intolerance secondary to atherosclerosis or collagen vascular disease. The results were analyzed using the criteria of ulcer healing and the prevention of amputation. In 6 of the 7 patients, the digital ulcers healed and amputation was avoided.

Bidirectional transepithelial water transport: measurement and governing mechanisms.

In the search for the mechanisms whereby water is transported across biological membranes, we hypothesized that in the airways, the hydration of the periciliary fluid layer is regulated by luminal-to-basolateral water transport coupled to active transepithelial sodium transport. The luminal-to-basolateral (JWL-->B) and the basolateral-to-luminal (JWB-->L) transepithelial water fluxes across ovine tracheal epithelia were measured simultaneously. The JWL-->B (6.1 microliter/min/cm2) was larger than JWB-->L (4.5 microliter/min/cm2, p < 0.05, n = 30). The corresponding water diffusional permeabilities were PdL-->B = 1.0 x 10(-4) cm/s and PdB-->L = 7.5 x 10(-5) cm/s. The activation energy (Ea) of JWL-->B (11.6 kcal/mol) was larger than the Ea of JWB-->L (6.5 kcal/mol, p < 0.05, n = 5). Acetylstrophanthidin (100 microM basolateral) reduced JWL-->B from 6.1 to 4.4 microliter/min/cm2 (p < 0. 05, n = 5) and abolished the PD. Amiloride (10 microM luminal) reduced JWL-->B from 5.7 to 3.7 microliter/min/cm2 (p < 0.05, n = 5) and reduced PD by 44%. Neither of these agents significantly changed JWB-->L. These data indicate that in tracheal epithelia under homeostatic conditions, JWB-->L was dominated by diffusion (Ea = 4.6 kcal/mol), whereas approximately 30% of JWL-->B was coupled to the active Na+,K+-ATPase pump (Ea = 27 kcal/mol).

Neuropeptide Y inhibits ciliary beat frequency in human ciliated cells via nPKC, independently of PKA.

The intracellular mechanisms whereby the inhibitory neurotransmitter neuropeptide Y (NPY) decreases ciliary beat frequency (CBF) were investigated in cultured human tracheal and bronchial ciliated cells. CBF was measured by nonstationary analysis laser light scattering. NPY at 1 and 10 microM decreased CBF from a baseline of 6.7 +/- 0.5 (n = 12) to 6.1 +/- 0.5 (P < 0.05) and 5.8 +/- 0.4 (P < 0.01) Hz, respectively. Prior application of PYX-1, an NPY antagonist, prevented the decreases of CBF induced by both doses of NPY. Two broad protein kinase C (PKC) kinase inhibitors, staurosporine and calphostin C, also abolished the NPY-induced decrease in CBF. The NPY-induced decrease in CBF was abolished by GF 109203X, a novel PKC (nPKC) isoform inhibitor, whereas this decrease in CBF was not attenuated by Gö-6976, a specific inhibitor of conventional PKC isoforms. Because pretreatment with NPY did not block the stimulation of CBF by forskolin and pretreatment with forskolin did not abolish the NPY-induced inhibition of CBF, this NPY receptor-mediated signal transduction mechanism appears to be independent of the adenylate cyclase-protein kinase A (PKA) pathway. Inhibition of Ca2+-ATPase by thapsigargin also prevented the suppression of CBF induced by subsequent application of NPY. These novel data indicate that, in cultured human epithelia, NPY decreases CBF below its basal level via the activation of an nPKC isoform and Ca2+-ATPase, independent of the activity of PKA. This is consistent with the proposition that NPY is an autonomic efferent inhibitory neurotransmitter regulating mucociliary transport.

Fluorescence and laser photon counting: measurements of epithelial [Ca2+]i or [Na+]i with ciliary beat frequency.

We describe a system we developed that enabled simultaneous measurements of either epithelial calcium ion concentration ([Ca2+]i) or sodium ion concentration ([Na+]i) with the ciliary beat frequency (CBF) in native ciliated epithelia using either Fura-2 (AM) or SBFI (AM) ratiometric fluorescence photon counting along with nonstationary laser light scattering. Studies were performed using native epithelial tissues obtained from ovine tracheae. The dynamic range of the laser light-scattering system was determined by a simulated light "beating" experiment. The nonstationary CBF was demonstrated by the time-frequency analysis of the raw photon count sequences of backscattered heterodyne photons from cultured and native epithelia. Calibrations of calcium and sodium ion concentrations were performed using the respective Fura-2 and SBFI impermanent salts as well as in native epithelia. The cumulative responses of 10(-6), 10(-5), and 10(-4) M nifedipine on [Ca2+]i together with the CBF as well as the cumulative responses of 10(-5), 10(-4), and 10(-3) M amiloride on [Na+]i together with the CBF were also determined. Nifedipine decreased [Ca2+]i but had no effect on CBF. Amiloride decreased [Na+]i and CBF. Stimulation of CBF corresponded with either an increase of [Na+]i or an increase of [Ca2+]i. Decreases of [Na+]i or substantial decreases of [Ca2+]i were associated with decreases in the CBF. These data demonstrate the utility of this system for investigating the regulatory mechanisms of intracellular ions dynamics and the CBF in native epithelia.

Depolarization of cell membrane is associated with an increase in ciliary beat frequency (CBF).

We hypothesize that activation of muscarinic cholinergic receptors depolarizes the cell membrane of the mammalian ciliated cells which in turn causes an increase of CBF. To test this hypothesis, a di-8-ANEPPS fluorescence photon counting and nonstationary heterodyne laser light scattering system was developed to measure cell membrane potential (psi) and CBF in cultured ovine tracheal ciliated cells simultaneously. Carbachol dose dependently depolarized the cell membrane with a corresponding stimulation of CBF. The carbachol induced depolarization of cell membrane and increases of CBF were inhibited by prior application of either atropine or verapamil or amiloride. These novel data suggest that depolarization of the cell membrane and the corresponding stimulation of CBF caused by the activation of muscarinic receptors of the mammalian ciliated cells are dependent on the influx of either extracellular Ca2+ or Na+.

Capsaicin-activated bronchial- and alveolar-initiated pathways regulating tracheal ciliary beat frequency.

We questioned whether the prolonged stimulation of ciliary beat frequency (CBF) to a short exposure of low-dose capsaicin (Wong et al. J. Appl. Physiol. 68: 257-2580, 1990) could be due to the activation of indirect pathways involving neural reflexes initiated independently in the bronchi and alveoli. Tracheal CBF (CBFtr) was measured temporally in anesthetized groups of 10 dogs by means of heterodyne-mode correlation analysis laser light scattering. To elucidate the site of the afferent neural stimulation and the efferent mediators affecting the ciliated epithelium, capsaicin (3 nM) aerosol was delivered for 4 min, either predominantly to the bronchi or to the alveolar regions, with use of pulsed aerosol techniques. This resulted in 13 pg of bronchial (85%) and 10 pg of alveolar (96%) capsaicin deposited, which caused marked stimulation of CBFtr with maxima at 7 and 35 min, respectively. Prior administration of aerosolized indomethacin to the bronchi or aerosolized cromolyn to the alveoli inhibited the bronchial and alveolar responses, respectively. Prior administration of aerosolized hexamethonium to the tracheal lumen blocked the stimulatory CBFtr responses from both capsaicin challenges. Ipratropium or propranolol aerosols delivered to the tracheal lumen also inhibited these responses. It is proposed that these pathways comprise one set of sensitive mechanisms to ensure a prolonged stimulation of CBF to effect the removal of secretions and the irritant from the lungs.

Stationary and nonstationary correlation-frequency analysis of heterodyne mode laser light scattering: magnitude and periodicity of canine tracheal ciliary beat frequency in vivo.

Stationary and nonstationary correlation-frequency analysis of heterodyne laser light scattering were utilized to make automated, on-line, objective measurements of tracheal ciliary beat frequency (CBF) in intact, anesthetized canines. The stationary correlation-frequency analysis laser light-scattering technique was used to assess the magnitude of the CBF stimulatory responses induced by aerosolized 10(-5) M fenoterol (sympathomimetic), and 10(-8) M and 10(-6) M methacholine (parasympathomimetic) delivered to the whole lungs of eight barbiturate-anesthetized beagles. The nonstationary correlation-frequency analysis laser light-scattering technique was used to measure the effect on tracheal CBF of increasing the cytosolic calcium ion concentration with a calcium ionophore, A23187. Aerosolized A23187 was delivered to the isolated tracheal lumens of eight beagle dogs in cumulative doses ranging from 10(-9)M to 10(-6) M. Administration of the ionophore synchronized the CBF with a period of 5.3 min. Dose dependencies were observed in both the time to the peak CBF stimulation and the magnitude of the stimulatory response. The magnitude of CBF stimulation was inhibited by prior administration of aerosolized nifedipine (2 mg/ml), a voltage-operated calcium channel blocker. The A23187-induced modulation period of tracheal CBF, was unchanged by nifedipine. These are the first data to demonstrate that the magnitude and periodicity of CBF are two independent coupled processes. The cooperativity of these two processes could be determined in the effectiveness of mucociliary transport.

Nature of the mammalian ciliary metachronal wave.

The temporal and spatial coordination of ciliary beat (metachronicity) is fundamental to effective mucociliary transport. Metachronal wave period (MWP) and ciliary beat frequency (CBF) of fresh excised sheep and canine tracheal epithelial tissues were measured with the use of a newly developed alternating focal spot laser light scattering system. MWP was determined from cross correlation of the heterodyne signals from the alternating focal spots. CBF was determined by autocorrelation of the heterodyne signals from each of the spots. MWP and CBF were measured in four sheep tracheal epithelial tissues with the use of longitudinal interfocal spot distances of 6 and 18 microns. In three canine tracheal epithelial tissues MWP and CBF were measured both longitudinally and circumferentially with interfocal spot distances of 5, 15, 65, 87, and 96 microns. For the sheep tracheal epithelial tissues the mean CBF was 5.9 +/- 0.4 Hz (mean of means; range 3.6 +/- 0.5 to 9.9 +/- 1.5 Hz), whereas the mean MWPs for 6- and 18-microns interfocal spot distances were 0.50 +/- 0.1 and 0.47 +/- 0.1 s, respectively. For the canine tracheal epithelial tissues the mean CBF was 4.0 +/- 0.2 Hz (2.0 +/- 0.8 to 7.2 +/- 3.2 Hz), whereas the mean longitudinal MWP was 1.5 s and the mean circumferential MWP was 2.1 s. Geometric combination of the MWP components leads to a derived MWP of 2.6 s with a propagation direction of 54 degrees with respect to the longitudinal axis of the trachea. MWP was found to be episode modulated with 12- to 20-min intervals in the longitudinal direction, but modulation was not as apparent in the circumferential direction. These data suggest that MWP and CBF are regulated by separate intracellular, intercellular, and intraciliary mechanisms.

Luminal purinergic regulatory mechanisms of tracheal ciliary beat frequency.

To investigate the modulation of tracheal ciliary beat frequency (CBFt) by purine nucleotides and nucleosides acting on luminal receptors, aerosolized ATP, GTP, AMP-PNP, GMP-PNP, adenosine, and guanosine were each administered separately to the tracheal lumen in eucapnically ventilated, barbiturate-anesthetized beagles. Four studies were conducted in each of seven dogs from a cohort of eight dogs. The CBFt responses were measured on the right lateral surface of the mid-trachea using heterodyne mode correlation analysis laser light scattering. Aerosolized 10(-6) M and 10(-5) M ATP stimulated CBFt from the baseline of 5.9 +/- 1.4 Hz to maxima of 12.1 +/- 1.4 Hz and 13.3 +/- 1.6 Hz, respectively, while the same corresponding ATP-analogue (AMP-PNP) concentrations stimulated baseline CBFt to maxima of 12.7 +/- 4.1 Hz and 18.1 +/- 2.1 Hz, respectively. Similarly, 10(-6) M and 10(-5) M GTP stimulated baseline CBFt to maxima of 14.8 +/- 1.1 Hz and 12.8 +/- 4.6 Hz, respectively. The corresponding GTP-analogue (GMP-PNP) concentrations stimulated CBFt to maxima of 14.5 +/- 2.1 Hz and 18.8 +/- 4.4 Hz, respectively. Prior delivery of 10(-5) M adenosine reduced all these nucleotide-induced stimulatory responses. Prior delivery of 10(-5) guanosine partially reduced the GTP- and the GMP-PNP-induced stimulatory responses. These data demonstrate that nucleotides and nucleosides modulate CBFt through specific P2 and P1 purinergic receptors on the luminal surface, thus providing a direct mechanism within the airways to enhance the transport of mucus.

Short-term interaction of airway and tissue oxygen tensions on ciliary beat frequency in dogs.

The responses of canine tracheal ciliary beat frequency (CBFt) to total lung, tracheal lumen, and peripheral lung hyperoxia, to tracheal lumen anoxia with or without peripheral lung hypoxia, and to isolated tracheal lumen hyperoxia combined with a beta-antagonist were delineated in anesthetized beagle dogs. CBFt was measured using a heterodyne laser light-scattering technique. When oxygen mixtures were delivered to the whole lung, a dose-dependent increase in maximal CBFt was observed from 6.3 +/- 1.0 Hz on air to 13.8 +/- 1.2 Hz on 100% oxygen. When oxygen mixtures were delivered to the isolated tracheal lumen, a dose-dependent increase in maximal CBFt from 6.9 +/- 1.3 Hz on air to 25.7 +/- 6.3 Hz on 100% oxygen was observed. CBFt was unchanged under conditions of peripheral lung hyperoxia. There were no significant changes from room air baseline CBFt of 7.6 +/- 1.5 Hz due to either isolated tracheal anoxia alone or in combination with alveolar hypoxia. CBFt stimulated with 100% oxygen insuffiated to the isolated tracheal lumen decreased from 14.1 +/- 3.2 to 9.5 +/- 1.9 Hz and from 16.5 +/- 1.2 to 9.7 +/- 1.2 Hz in response to 6 and 18 micrograms/kg of intravenous esmolol, respectively. This study demonstrates that short-term, local hyperoxia stimulates CBFt and that a pulmonary or systemically derived factor can be activated to inhibit this stimulation. It indicates that acute airway anoxia and alveolar and blood hypoxia do not suppress ciliary beat frequency. It also suggests that the adrenergic system is involved in the oxygen-induced stimulation of ciliary beat.

Tracheal ciliary beat frequency in baboons: effects of peripheral histamine and capsaicin.

To determine if tracheal ciliary beat frequency (CBFt) in the nonhuman primate could be stimulated indirectly, either by an inhaled irritant or by a mediator potentially released from an inhalation challenge, saline (0.9%), capsaicin (3 x 10(-9) M), or histamine (8 mg/ml) were delivered for three minutes as 100 ml aerosol (0.25 micron MMAD) boluses at the beginning of each breath, followed by 10 sec breath hold. Each of five adult baboons (17-27 kg, Papio cynocephalus) underwent three studies using a randomized block design. The baboons were anesthetized and ventilated eucapnically through an endotracheal tube whose cuff was inflated in the distal trachea. CBFt was measured in the mid-trachea proximal to the cuff at one minute intervals using heterodyne laser light scattering. There was no change in CBFt from a baseline of 7.2 +/- 0.6 Hz (mean +/- SE) following inhalation of saline deposited peripherally. Capsaicin stimulated CBFt for over 80 min reaching a maximum of 14.7 +/- 6.3 Hz 46 min after aerosol delivery. Histamine stimulated CBFt for 90 minutes reaching a maximum of 19.3 +/- 9.9 Hz 67 min after aerosol delivery. The prolonged stimulatory responses of CBFt to peripherally deposited capsaicin and histamine most likely involve both cellular and neural pathways through which the tracheobronchial airways, in response to an alveolar challenge, are cleansed of secretions thus enabling the maintenance of effective gas transport.

Pathways of substance P stimulation of canine tracheal ciliary beat frequency.

Substance P (SP), an inflammatory neuropeptide, may be released by intraepithelial nerves in response to an irritant or inflammatory stimulus. To investigate the neural and humoral pathways mediating the response of tracheal ciliary beat frequency (CBF) to topically applied SP, CBF was measured on the ventral midtracheal surface of anesthetized beagles by using heterodyne-mode correlation analysis laser light scattering. In the first study, aerosolized SP, delivered to the lungs of eight beagle dogs, stimulated CBF in a dose-dependent manner from a baseline of 4.9 +/- 0.4 Hz to a maximum of 14.9 +/- 1.5 Hz at dose of 10(-7) M. In the second study, the tracheal lumen was isolated from the bronchial airways by inflating the cuff of an endotracheal tube near the carina. Intravenous hexamethonium bromide (2 mg/kg), ipratropium bromide (0.5 micrograms/kg), and indomethacin (2 mg/kg) were used as blocking agents to inhibit the nicotinic, muscarinic, and cyclooxygenase pathways, respectively. Aerosolized 10(-9), 10(-8), or 10(-7) M SP was delivered sequentially to the tracheal lumen for 3 min at 30-min intervals. SP caused two distinct CBF stimulatory episodes at 4 min (mean time of the maximal response) and at 18 min (mean time of the maximal response) after onset of delivery and returned to baseline after 25 min. SP stimulated CBF from the baseline of 5.1 +/- 0.4 Hz to a maximum of 14.2 +/- 2.5 Hz during the first episode (P less than 0.01) and to 10.4 +/- 0.6 Hz during the second episode (P less than 0.01) at dose of 10(-8) M. These responses were inhibited by all the blocking agents. These data suggest that SP stimulates CBF via a cyclooxygenase-dependent parasympathetic reflex.

Stimulation of tracheal ciliary beat frequency by capsaicin.

To determine the possible involvement of neural and cyclooxygenase pathways whereby irritants might affect cilia activity in vivo, the temporal response of canine tracheal ciliary beat frequency (CBF) to the inhaled surrogate irritant capsaicin was studied. CBF was measured on the ventral midtracheal surface of barbiturate-anesthetized eucapnically ventilated beagle dogs by heterodyne-mode laser light scattering. After base-line CBF was established, hexamethonium bromide (2 mg/kg iv), ipratropium bromide (0.5 microgram/kg iv), indomethacin (2 mg/kg iv), or intravenous 0.9% saline was administered. Aerosolized 3 Z 10(-9) M capsaicin in 0.9% saline was delivered for 2 min, and CBF was measured for the following 60 min. Control experiments used 0.9% saline sham aerosol with a 0.9% saline sham block. Aerosolized capsaicin stimulated CBF from a base line of 6.2 +/- 1.4 (SD) Hz (n = 230) to a mean maximum of 17.7 +/- 7.3 Hz (n = 16) 23 min after aerosol delivery, and CBF returned to base line within 60 min. Neither hexamethonium bromide, ipratropium bromide, nor indomethacin changed CBF from base-line values. The episodic CBF stimulatory response to capsaicin after commencement of aerosol was completely inhibited by hexamethonium bromide. Ipratropium bromide partially inhibited the first 15 min and totally inhibited the following 45 min of stimulatory response. Indomethacin inhibited the initial 15 min but had less effect on the following 45 min of stimulatory response. These data indicate that multiple stimulatory mechanisms function over a prolonged period of time to affect the removal of irritants from the airways and that these mechanisms differ from those involved in the maintenance of basal CBF.

Regulatory pathways for the stimulation of canine tracheal ciliary beat frequency by bradykinin.

1. The effects of bradykinin, a potent inflammatory nanopeptide, on tracheal ciliary beat frequency in vivo were investigated using barbiturate-anaesthetized beagles. Tracheal ciliary beat frequency was measured using heterodyne mode correlation analysis laser light scattering, a technique that does not require surgical intervention. 2. Aerosolized 10(-5) M-bradykinin in 0.9% saline administered for 3 min to eight barbiturate-anaesthetized beagles stimulated tracheal ciliary beat frequency from the baseline of 5.3 +/- 0.1 Hz to a maximum of 16.6 +/- 2.0 Hz, 8 min after aerosol delivery, and ciliary beat frequency remained above baseline for the following 35 min. 3. Intravenously injected hexamethonium bromide, ipratropium bromide or indomethacin did not change baseline tracheal ciliary beat frequency. That down-regulation of ciliary beat frequency below baseline values was not observed with either the neural or the cyclooxygenase blocking agents suggests that neither of these pathways is involved in the maintenance of the observed basal ciliary beat frequency. 4. Bradykinin-induced stimulation of tracheal ciliary beat frequency is blocked by hexamethonium bromide, ipratropium bromide or indomethacin. These data suggest that the stimulation of ciliary beat frequency by bradykinin acts through both cellular cyclooxygenase and parasympathetic pathways in series.

Stimulation of tracheal ciliary beat frequency by localized tissue incision.

Since measurements of basal ciliary beat frequency (CBF) were significantly lower in our intact canine experiments than reports of ciliary activity in rabbits involving surgical intervention, we hypothesized that local tissue trauma stimulates CBF. The effects of minor neck surgery on tracheal CBF in eight barbiturate-anesthetized eucapnically ventilated beagles were investigated. Each dog underwent two studies. Measurements of CBF were made at 1-min intervals on the right lateral midtracheal surface by means of heterodyne mode correlation analysis laser light scattering. In the control study, CBF was measured in each dog for at least 160 min. In the incision study, base-line CBF was measured for at least 40 min. The overlying sternohyoidus muscles were then separated, and a longitudinal 2- to 3-cm incision was made in the trachea caudally from the fourth to the fifth cartilage ring. CBF was measured at least 5 cm distally from the site of tracheal injury for an additional 120 min. Electrocardiogram, rectal temperature, tracheal pressure, exhaled CO2, and arterial blood pressure, PO2, PCO2, and pH remained stable throughout both studies. The mean base-line CBF was 4.7 +/- 0.4 Hz. It increased to 19.5 +/- 2.9 Hz (P less than 0.0001) 100 min after the incision and remained elevated until the end of the study period (P less than 0.0001). The mechanism(s) causing this stimulation may also be responsible for the high "basal" CBF observed in other studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of ciliary beat frequency by autonomic mechanisms: in vitro.

The ciliated epithelium of the mammalian trachea separates the neurohumoral milieu of the tissue from that of the environment of the airway lumen. To determine whether specific autonomic receptors regulating ciliary beat frequency (CBF) were located on mucosal or serosal sides, we measured CBF by heterodyne mode correlation analysis laser light scattering in bovine tracheal tissues mounted in a two-sided chamber. A beta 2-adrenergic agonist, fenoterol, at 10(-7) M, stimulated serosal CBF from 7.9 +/- 1.3 to 20.2 +/- 5.8 Hz (P less than 0.01) and mucosal CBF from 6.6 +/- 0.9 to 14.7 +/- 4.6 Hz (P less than 0.01). A muscarinic cholinergic agonist, methacholine, at 10(-7) M, increased mucosal CBF from 8.4 +/- 1.0 to 19.5 +/- 5.5 Hz (P less than 0.01) and serosal CBF from 8.0 +/- 0.9 to 15.4 +/- 5.0 Hz (P less than 0.01). The differences in stimulation of CBF on the mucosal and serosal sides between fenoterol and methacholine were significant (P less than 0.01). Studies in which these autonomic agonist stimulating effects were inhibited by their respective antagonists, propranolol and atropine sulfate, demonstrated that CBF can be regulated independently by mediators both in the submucosa and within the mucus lining.