First-in-Human Clinical Trial to Assess the Safety, Tolerability and Pharmacokinetics of Single Doses of NTM-1633, a Novel Mixture of Monoclonal Antibodies against Botulinum Toxin E.

Botulism is a rare, life-threatening paralytic disease caused by botulinum neurotoxin (BoNT). Available treatments including an equine antitoxin and human immune globulin are given postexposure and challenging to produce and administer. NTM-1633 is an equimolar mixture of 3 human IgG monoclonal antibodies, E1, E2, and E3, targeting BoNT serotype E (BoNT/E). This first-in-human study assessed the safety, tolerability, pharmacokinetics (PK), and immunogenicity of NTM-1633. This double-blind, single-center, placebo-controlled dose escalation study randomized 3 cohorts of healthy volunteers to receive a single intravenous dose of NTM-1633 (0.033, 0.165, or 0.330 mg/kg) or saline placebo. Safety monitoring included physical examinations, clinical laboratory studies, and vital signs. Blood sampling was performed at prespecified time points for PK and immunogenicity analyses. Twenty-four subjects received study product (18 NTM-1633; 6 placebo), and no deaths were reported. An unrelated serious adverse event was reported in a placebo subject. Adverse events in the NTM-1633 groups were generally mild and similar in frequency and severity to the placebo group, and no safety signal was identified. NTM-1633 has a favorable PK profile with a half-life >10 days for the 0.330 mg/kg dose and an approximately linear relationship with respect to maximum concentration and area under the concentration-time curve (AUC0→t). NTM-1633 also demonstrated low immunogenicity. NTM-1633 is well tolerated at the administered doses. The favorable safety, PK, and immunogenicity profile supports further development as a treatment for BoNT/E intoxication and postexposure prophylaxis.

Publisher's Note: Effects of Lifshitz Transitions on Charge Transport in Magnetic Phases of Fe-Based Superconductors [Phys. Rev. Lett. 114, 097003 (2015)].

This corrects the article DOI: 10.1103/PhysRevLett.114.097003.

Autonomic nervous system function assessed by conventional and spectral analysis might be useful in terms of predicting retinal deterioration in persons with type 1 diabetes mellitus.

AIMS: To determine whether cardiac autonomic dysfunction represents a risk factor for diabetic retinopathy (DR) development and progression in persons with type 1 diabetes mellitus (T1DM). METHODS: The study comprised 154 normoalbuminuric persons with T1DM divided into two groups according to the DR presence: with and without DR. Cardiovascular autonomic functioning was measured at baseline using conventional and spectral analysis. Participants were re-examined for the DR presence 18months after. RESULTS: The group with DR had longer disease duration compared to the group without DR (20 vrs 11.5years, p<0.001), heart rate coefficient of variation (HRV-CV) at rest and during deep breathing were lower in participants with DR (p=0.001 and 0.004), as well did spectral indices of HRV: low frequency (LF) band, high frequency (HF) band (p=0.003 and 0.022) while LF/HF ratio indicating sympathovagal balance was higher (p=0.037). No difference in glycaemic control or blood pressure value were observed. Twenty-one (13.36%) participants developed non proliferative DR or progressed to proliferative DR. Cox proportional regression showed that the 18months risk from retinal deterioration was reduced by 33.4% by each increase in the HRV-CV of 1%, 12.7% for the same HRV-CV increase during deep breathing while LF band of 1ms(2) results in 8.6% risk reduction. CONCLUSIONS: This study provides evidence that DR should not be considered merely a metabolic control manifestation and that HRV-CV as well as spectral indices of HRV might serve as a practical tool to identify a subgroup of T1DM patients with higher risk of retinal deterioration.

Effects of Lifshitz transition on charge transport in magnetic phases of Fe-based superconductors.

The unusual temperature dependence of the resistivity and its in-plane anisotropy observed in the Fe-based superconducting materials, particularly Ba(Fe_{1-x}Co_{x})_{2}As_{2}, has been a long-standing puzzle. Here, we consider the effect of impurity scattering on the temperature dependence of the average resistivity within a simple two-band model of a dirty spin density wave metal. The sharp drop in resistivity below the Néel temperature T_{N} in the parent compound can only be understood in terms of a Lifshitz transition following Fermi surface reconstruction upon magnetic ordering. We show that the observed resistivity anisotropy in this phase, arising from nematic defect structures, is affected by the Lifshitz transition as well.

Safety and pharmacokinetics of XOMA 3AB, a novel mixture of three monoclonal antibodies against botulinum toxin A.

Botulinum neurotoxin A is a category A bioterrorism agent. Current antitoxin therapies are scarce and produce adverse reactions. XOMA 3AB consists of 3 IgG1 monoclonal antibodies (MAbs), each with a distinct human or humanized variable region, which bind to distinct epitopes on botulinum neurotoxin serotype A. This first-in-human study evaluated the safety and pharmacokinetics (PK) of escalating doses of XOMA 3AB administered intravenously (i.v.) to healthy adults. In this double-blind placebo-controlled dose escalation study, 3 cohorts of 8 healthy subjects received a single intravenous dose of XOMA 3AB or placebo at a 3:1 ratio. Follow-up examinations included physical examinations, hematology and chemistry blood tests, electrocardiograms, and pharmacokinetics. Pharmacokinetic parameters were estimated using noncompartmental methods. There were no infusion discontinuations or hypersensitivity reactions. Two or more subjects experienced headache, hyperglycemia, or anemia; none was dose related. All adverse events (AEs) were mild to moderate except for an episode of exercise-induced elevation of a subject's creatine phosphokinase (CPK) level, unrelated to XOMA 3AB. Concentration-time plots demonstrated a peak in MAb concentrations 1 to 2 h after completion of the infusion, after which the levels declined in a biexponential decay pattern for all analytes. For each MAb, the maximum concentration of drug in serum (Cmax) and the area under the concentration-time curve from 0 to infinity (AUCinf) increased as the dose increased. Clearance of the humanized mouse MAb was more rapid than that of the two fully human MAbs, particularly at the lowest dose. None of the MAbs was immunogenic. At the doses administered, XOMA 3AB was well tolerated. These safety findings support further investigation of XOMA 3AB as a potential agent for botulism treatment and postexposure prophylaxis. (This study has been registered at ClinicalTrials.gov under registration no. NCT01357213.).

Engineered domain-based assays to identify individual antibodies in oligoclonal combinations targeting the same protein.

Quantitation of individual monoclonal antibodies (mAbs) within a combined antibody drug product is required for preclinical and clinical drug development. We have developed two antitoxins, XOMA 3B and XOMA 3E, each consisting of three mAbs that neutralize type B and type E botulinum neurotoxin (BoNT/B and BoNT/E) to treat serotype B and E botulism. To develop mAb-specific binding assays for each antitoxin, we mapped the epitopes of the six mAbs. Each mAb bound an epitope on either the BoNT light chain (LC) or translocation domain (H(N)). Epitope mapping data were used to design LC-H(N) domains with orthogonal mutations to make them specific for only one mAb in either XOMA 3B or XOMA 3E. Mutant LC-H(N) domains were cloned, expressed, and purified from Escherichia coli. Each mAb bound only to its specific domain with affinity comparable to the binding to holotoxin. Further engineering of domains allowed construction of enzyme-linked immunosorbent assays (ELISAs) that could characterize the integrity, binding affinity, and identity of each of the six mAbs in XOMA 3B and 3E without interference from the three BoNT/A mAbs in XOMA 3AB. Such antigen engineering is a general method allowing quantitation and characterization of individual mAbs in a mAb cocktail that bind the same protein.

Dependence of the excitability of pituitary cells on cyclic nucleotides.

Cyclic 3',5'-adenosine monophosphate and cyclic 3',5'-guanosine monophosphate are intracellular (second) messengers that are produced from the nucleotide triphosphates by a family of enzymes consisting of adenylyl and guanylyl cyclases. These enzymes are involved in a broad array of signal transduction pathways mediated by the cyclic nucleotide monophosphates and their kinases, which control multiple aspects of cell function through the phosphorylation of protein substrates. We review the findings and working hypotheses on the role of the cyclic nucleotides and their kinases in the control of electrical activity of the endocrine pituitary cells and the plasma membrane channels involved in this process.

Dependence of spontaneous electrical activity and basal prolactin release on nonselective cation channels in pituitary lactotrophs.

All secretory anterior pituitary cells fire action potentials spontaneously and exhibit a high resting cation conductance, but the channels involved in the background permeability have not been identified. In cultured lactotrophs and immortalized GH(3) cells, replacement of extracellular Na(+) with large organic cations, but not blockade of voltage-gated Na(+) influx, led to an instantaneous hyperpolarization of cell membranes that was associated with a cessation of spontaneous firing. When cells were clamped at -50 mV, which was close to the resting membrane potential in these cells, replacement of bath Na(+) with organic cations resulted in an outward-like current, reflecting an inhibition of the inward holding membrane current and indicating loss of a background-depolarizing conductance. Quantitative RT-PCR analysis revealed the high expression of mRNA transcripts for TRPC1 and much lower expression of TRPC6 in both lactotrophs and GH(3) cells. Very low expression of TRPC3, TRPC4, and TRPC5 mRNA transcripts were also present in pituitary but not GH(3) cells. 2-APB and SKF-96365, relatively selective blockers of TRPC channels, inhibited electrical activity, Ca(2+) influx and prolactin release in a concentration-dependent manner. Gd(3+), a common Ca(2+) channel blocker, and flufenamic acid, an inhibitor of non-selective cation channels, also inhibited electrical activity, Ca(2+) influx and prolactin release. These results indicate that nonselective cation channels, presumably belonging to the TRPC family, contribute to the background depolarizing conductance and firing of action potentials with consequent contribution to Ca(2+) influx and hormone release in lactotrophs and GH(3) cells.

Domain-based assays of individual antibody concentrations in an oligoclonal combination targeting a single protein.

Quantitation of individual monoclonal antibodies (mAbs) within a combined antibody drug product is required for preclinical and clinical drug development, including pharmacokinetic (PK), toxicology, stability, and biochemical characterization studies of such drugs. We have developed an antitoxin, XOMA 3AB, consisting of three recombinant mAbs that potently neutralize the known subtypes of type A botulinum neurotoxin (BoNT/A). The three mAbs bind nonoverlapping BoNT/A epitopes with high affinity. XOMA 3AB is being developed as a treatment for botulism resulting from BoNT/A. To develop antibody-specific assays, we cloned, expressed, and purified BoNT/A domains from Escherichia coli. Each mAb bound only to its specific domain with affinity comparable to the binding to holotoxin. mAb-specific domains were used to develop an enzyme-linked immunosorbent assay (ELISA) for characterization of the integrity and binding activity of the three mAbs in the drug product. An electrochemiluminescence bridging assay that is robust to interference from components in serum was also developed, and we demonstrate that it can be used for PK assays. This type of antigen engineering to generate mAb-specific domains is a general method allowing quantitation and characterization of individual mAbs in a mAb cocktail that binds the same protein and is superior to anti-idiotype approaches.

Neutralizing human monoclonal antibodies binding multiple serotypes of botulinum neurotoxin.

Botulism, a disease of humans characterized by prolonged paralysis, is caused by botulinum neurotoxins (BoNTs), the most poisonous substances known. There are seven serotypes of BoNT (A-G) which differ from each other by 34-64% at the amino acid level. Each serotype is uniquely recognized by polyclonal antibodies, which originally were used to classify serotypes. To determine if there existed monoclonal antibodies (mAbs) capable of binding two or more serotypes, we evaluated the ability of 35 yeast-displayed single-chain variable fragment antibodies generated from vaccinated humans or mice for their ability to bind multiple BoNT serotypes. Two such clonally related human mAbs (1B18 and 4E17) were identified that bound BoNT serotype A (BoNT/A) and B or BoNT/A, B, E and F, respectively, with high affinity. Using molecular evolution techniques, it proved possible to both increase affinity and maintain cross-serotype reactivity for the 4E17 mAb. Both 1B18 and 4E17 bound to a relatively conserved epitope at the tip of the BoNT translocation domain. Immunoglobulin G constructed from affinity matured variants of 1B18 and 4E17 were evaluated for their ability to neutralize BoNT/B and E, respectively, in vivo. Both antibodies potently neutralized BoNT in vivo demonstrating that this epitope is functionally important in the intoxication pathway. Such cross-serotype binding and neutralizing mAbs should simplify the development of antibody-based BoNT diagnostics and therapeutics.

Alzheimer's disease: more than amyloid.

CONTEXT: The etiology of Alzheimer's disease (AD) is inconclusive. Treatments targeting amyloid have largely been unsuccessful. There is increasing evidence that vasculopathy may play an important pathogenic role in AD. OBJECTIVE: Longitudinal measurements of whole blood viscosity (WBV) using a computer-assisted hemorheologic protocol and characterization of microvascular abnormalities using computer-assisted intravital microscopy (CAIM) are two objective methods adopted in this laboratory to noninvasively quantify vasculopathy in AD patients. A correlation of increased disease severity with worsened vasculopathy would further bolster a cause and effect relationship. A case report (Case 1) is presented to illustrate the usefulness of following an AD patient with these noninvasive techniques to correlate disease progression with vasculopathy. DESIGN: Patients were selected from a private practice setting who met the Diagnostic and Statistical Manual of Mental Disorders criteria for AD. The Rheolog™, a computer-assisted scanning rheometer, was used to obtain longitudinal measurements of WBV. The microvascular abnormalities in the bulbar conjunctiva were quantified using a severity index (SI, scale 0-15). The patient was observed over a 4 year period from 2005 to 2008. CONCLUSION: This case study shows a correlation of disease progression in an AD patient with worsened vasculopathy. It illustrates the usefulness of WBV and CAIM as tools to quantify vasculopathy in AD patients and additionally suggests a pathogenetic role vasculopathy may play in concert with the amyloid hypothesis.

The antihyperalgesic effect of levetiracetam in an inflammatory model of pain in rats: mechanism of action.

BACKGROUND AND PURPOSE: Levetiracetam, a novel antiepileptic drug, has recently been shown to have antinociceptive effects in various animal models of pain. The purpose of this study was to investigate the antihyperalgesic effect of levetiracetam and its mechanism of action, by examining the involvement of GABAergic, opioidergic, 5-hydroxytryptaminergic (5-HTergic) and adrenergic systems in its effect, in a rat model of inflammatory pain. EXPERIMENTAL APPROACH: Rats were intraplantarly injected with the pro-inflammatory compound carrageenan. A paw pressure test was used to determine: (i) the effect of levetiracetam on carrageenan-induced hyperalgesia; and (ii) the effects of bicuculline (selective GABA(A) receptor antagonist), naloxone (non-selective opioid receptor antagonist), methysergide (non-selective 5-HT receptor antagonist) and yohimbine (selective alpha(2)-adrenoceptor antagonist) on the antihyperalgesic action of levetiracetam. RESULTS: Levetiracetam (10-200 mg.kg(-1); p.o.) significantly reduced, in a dose-dependent manner, the inflammatory hyperalgesia induced by carrageenan. The antihyperalgesic effect of levetiracetam was significantly decreased after administration of bicuculline (0.5-2 mg.kg(-1); i.p.), naloxone (1-3 mg.kg(-1); i.p.), methysergide (0.25-1 mg.kg(-1); i.p.) and yohimbine (1-3 mg.kg(-1); i.p.). CONCLUSIONS AND IMPLICATIONS: These results show that levetiracetam produced antihyperalgesia which is at least in part mediated by GABA(A), opioid, 5-HT and alpha(2)-adrenergic receptors, in an inflammatory model of pain. The efficacy of levetiracetam in this animal model of inflammatory pain suggests that it could be a potentially important agent for treating inflammatory pain conditions in humans.

Correlation of microvascular abnormalities and endothelial dysfunction in Type-1 Diabetes Mellitus (T1DM): a real-time intravital microscopy study.

We hypothesize that real-time in vivo microvascular abnormalities should correlate with biochemical markers of inflammation/endothelial dysfunction in T1DM. Real-time quantification of T1DM and healthy non-diabetic control microcirculation was conducted utilizing computer-assisted intravital microscopy. Selected biochemical markers (high sensitivity C-reactive protein (hsCRP), soluble vascular cell adhesion molecules (sVCAM), soluble intercellular adhesion molecules (sICAM), soluble E-selectin (sE-selectin), nitrotyrosine, superoxide anion (O2-), interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha)) were used for correlation. The severity of microvascular abnormalities, as reflected by the arithmetic severity index (SI), was significantly increased in T1DM vs. controls (5.89 +/- 1.47 vs. 2.34 +/- 1.48; P<0.001). In addition several of the specific microvascular abnormalities (related to flow and morphometry) were significantly more prevalent in the T1DM patients. Finally, the following significant positive correlations existed between the inflammatory/endothelial dysfunction markers and specific microvascular abnormalities: sVCAM and abnormal vessel diameter (P=0.004, OR =1.033, 95% CI for OR =(1.01, 1.056)), superoxide (O2-) release and abnormal vessel distribution (P=0.032, OR =1.798, 95% CI for OR =(1.051, 3.075)), and sE-selectin and abnormal vessel distribution (P=0.036, OR =1.118, 95% CI for OR =(1.007, 1.241)). In view of such significant correlations, we conclude that these specific microvascular abnormalities can serve as unique physiologic markers of endothelial dysfunction to correlate with the biochemical markers of inflammatory/endothelial dysfunction in disease progression and therapeutic efficacy studies.

Opioidergic mechanisms are not involved in the antihyperalgesic effects of carbamazepine and oxcarbazepine.

The mechanisms of the analgesic action of carbamazepine and oxcarbazepine, in particular the role of opioid receptors, have not been established precisely. The systemic effects of naloxone, an opioid receptor antagonist, on the antihyperalgesic effects of carbamazepine and oxcarbazepine were examined in the model of inflammatory hyperalgesia induced by the intraplantar (i.pl.) administration of concanavaline A (Con A, 0.8 mg/paw) into the rat hind paw. Naloxone (3 mg/kg; i.p.) did not alter the antihyperalgesic effects of either carbamazepine or oxcarbazepine. These results indicate that the opioid system of pain modulation does not play a significant role in the antihyperalgesic effects of carbamazepine and oxcarbazepine.

Role of alpha2-adrenoceptors in the local peripheral antinociception by carbamazepine in a rat model of inflammatory mechanical hyperalgesia.

The anticonvulsant carbamazepine was recently shown to possess local peripheral antinociceptive properties. In this study, we investigated whether alpha2-adrenergic receptors are involved in the local peripheral antihyperalgesic effects of carbamazepine and determined the type of interaction between carbamazepine and clonidine, an alpha2-adrenoceptor agonist. Intraplantar (i.pl.) coadministration of either carbamazepine (100-1000 nmol/paw) or clonidine (1.9-3.7 nmol/paw) with the proinflammatory compound concanavalin A (Con A; 0.8 mg/paw) caused a significant dose- and time-dependent reduction of the difference between the forces exerted by a rat's hind paws in a modified paw-pressure test. The coadministration of 260 and 520 nmol/paw (i.pl.) yohimbine, an alpha2-adrenoceptor antagonist, with carbamazepine, significantly depressed the local antihyperalgesic effect in a dose- and time-dependent manner whereas yohimbine by itself did not have any effect. The administration of a mixture of carbamazepine and clonidine at fixed dose fractions (1/4, 1/2 and 3/4) of ED50 caused a significant and dose-dependent reduction of Con A-induced hyperalgesia. Isobolographic analysis revealed an additive interaction. These results suggest that alpha2-adrenoceptors play a role in the local peripheral antihyperalgesic effects of carbamazepine and that local peripheral coadministration of carbamazepine with clonidine results in an additive antihyperalgesic effect.

Peripheral anti-hyperalgesia by oxcarbazepine: involvement of adenosine A1 receptors.

In this study we determined whether oxcarbazepine (OXC) could produce local peripheral antinociceptive effects in a rat model of inflammatory hyperalgesia, and whether adenosine receptors were involved. When coadministered with the pro-inflammatory compound concanavalin A, OXC (1000-3000 nmol/paw) caused a significant dose- and time-dependent anti-hyperalgesia. Caffeine (1000-1500 nmol/paw), a nonselective adenosine receptor antagonist, as well as 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) (10-30 nmol/paw), a selective A1 receptor antagonist, coadministered with OXC, significantly depressed its anti-hyperalgesic effect. Drugs injected into the contralateral hind paw did not produce significant effects. These results indicate that OXC produces local peripheral anti-hyperalgesic effects, which is mediated via peripheral A1 receptors.

Dependence of hyperpolarisation-activated cyclic nucleotide-gated channel activity on basal cyclic adenosine monophosphate production in spontaneously firing GH3 cells.

The hyperpolarisation-activated cyclic nucleotide-gated (HCN) channels play a distinct role in the control of membrane excitability in spontaneously active cardiac and neuronal cells. Here, we studied the expression and role of HCN channels in pacemaking activity, Ca(2+) signalling, and prolactin secretion in GH(3) immortalised pituitary cells. Reverse transcriptase-polymerase chain reaction analysis revealed the presence of mRNA transcripts for HCN2, HCN3 and HCN4 subunits in these cells. A hyperpolarisation of the membrane potential below - 60 mV elicited a slowly activating voltage-dependent inward current (I(h)) in the majority of tested cells, with a half-maximal activation voltage of -89.9 +/- 4.2 mV and with a time constant of 1.4 +/- 0.2 s at -120 mV. The bath application of 1 mM Cs(+), a commonly used inorganic blocker of I(h), and 100 microM ZD7288, a specific organic blocker of I(h), inhibited I(h) by 90 +/- 4.1% and 84.3 +/- 1.8%, respectively. Receptor- and nonreceptor-mediated activation of adenylyl and soluble guanylyl cyclase and the addition of a membrane permeable cyclic adenosine monophosphate (cAMP) analogue, 8-Br-cAMP, did not affect I(h). Inhibition of basal adenylyl cyclase activity, but not basal soluble guanylyl cyclase activity, led to a reduction in the peak amplitude and a leftward shift in the activation curve of I(h) by 23.7 mV. The inhibition of the current was reversed by stimulation of adenylyl cyclase with forskolin and by the addition of 8-Br-cAMP, but not 8-Br-cGMP. Application of Cs(+) had no significant effect on the resting membrane potential or electrical activity, whereas ZD7288 exhibited complex and I(h)-independent effects on spontaneous electrical activity, Ca(2+) signalling, and prolactin release. These results indicate that HCN channels in GH(3) cells are under tonic activation by basal level of cAMP and are not critical for spontaneous firing of action potentials.

Effect of Helicobacter pylori eradication on extent of duodenal gastric metaplasia and grade of gastritis.

The extent of the regression of duodenal gastric metaplasia (DGM) after the eradication of Helicobacter pylori infection is controversial. Therefore, we decided to assess the degree of DGM before, sex weeks and one year after H. pylori eradication. 105 consecutive Helicobacter pylori positive patients with endoscopically proven duodenal ulcer, with DGM and Helicobacter pylori infection were recruited for this study. The diagnosis of Helicobacter pylori infection was based on CLO-test and histology, and DGM was assessed on four bulb biopsies taken before, sex weeks and one year after Helicobacter pylori eradication. Histological assessment of Helicobacter pylori associated gastritis was performed according to the Sydney classification. Follow up study on 98 patients before, six weeks and one year after the eradication of Helicobacter pylori showed that the mean extent of DGM did not change significantly after eradication and did not differ when compared with 14 patients with persisting infection. Our results show that the inflammatory process related to Helicobacter pylori does not play the main role in the development of DGM.

Regulation of Ca2+-sensitive adenylyl cyclase in gonadotropin-releasing hormone neurons.

In immortalized GnRH neurons, cAMP production is elevated by increased extracellular Ca2+ and the Ca2+ channel agonist, BK-8644, and is diminished by low extracellular Ca2+ and treatment with nifedipine, consistent with the expression of adenylyl cyclase type I (AC I). Potassium-induced depolarization of GT1-7 neurons causes a dose-dependent monotonic increase in [Ca2+]i and elicits a bell-shaped cAMP response. The inhibitory phase of the cAMP response is prevented by pertussis toxin (PTX), consistent with the activation of G(i)-related proteins during depolarization. Agonist activation of the endogenous GnRH receptor in GT1-7 neurons also elicits a bell-shaped change in cAMP production. The inhibitory action of high GnRH concentrations is prevented by PTX, indicating coupling of the GnRH receptors to G(i)-related proteins. The stimulation of cAMP production by activation of endogenous LH receptors is enhanced by low (nanomolar) concentrations of GnRH but is abolished by micromolar concentrations of GnRH, again in a PTX-sensitive manner. These findings indicate that GnRH neuronal cAMP production is maintained by Ca2+ entry through voltage-sensitive calcium channels, leading to activation of Ca2+-stimulated AC I. Furthermore, the Ca2+ influx-dependent activation of AC I acts in conjunction with AC-regulatory G proteins to determine basal and agonist-stimulated levels of cAMP production.