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Metabolism of opioid peptides by cerebral microvascular aminopeptidase M
Abstract:
Aminopeptidase M (EC 3.4.11.2), which can degrade low molecular weight opioid peptides, has been reported in both peripheral vasculature and in the CNS. Thus, we have studied the metabolism of opioid peptides by membrane-bound aminopeptidase M derived from cerebral microvessels of hog and rabbit. Both hog and rabbit microvessels were found to contain membrane-bound aminopeptidase M. At neutral pH, microvessels preferentially degraded low molecular weight opioid peptides by hydrolysis of the N-terminal Tyr1-Gly2 bond. Degradation was inhibited by amastatin (I50 = 0.2 microM) and bestatin (10 microM), but not by a number of other peptidase inhibitors including captopril and phosphoramidon. Rates of degradation were highest for the shorter peptides (Met5- and Leu5-enkephalin) whereas beta-endorphin was nearly completely resistant to N-terminal hydrolysis. Km values for the microvascular aminopeptidase also decreased significantly with increasing peptide length (Km = 91.3 +/- 4.9 and 28.9 +/- 3.5 microM for Met5-enkephalin and Met5-enkephalin-Arg6-Phe7, respectively). Peptides known to be present within or in close proximity to cerebral vessels (e.g., neurotensin and substance P) competitively inhibited enkephalin degradation (Ki = 20.4 +/- 2.5 and 7.9 +/- 1.6 microM, respectively). These data suggest that cerebral microvascular aminopeptidase M may play a role in vivo in modulating peptide-mediated local cerebral blood flow, and in preventing circulating enkephalins from crossing the blood-brain barrier.
Insights
Aminopeptidase M in cerebral microvessels degrades opioid peptides like enkephalins. This enzyme may regulate blood flow and prevent enkephalins from entering the brain.
Area of Science:
- Neuroscience
- Biochemistry
- Pharmacology
Background:
- Aminopeptidase M (APM) degrades opioid peptides and is found in the peripheral vasculature and central nervous system (CNS).
- Cerebral microvessels are crucial for regulating blood-brain barrier (BBB) function and cerebral blood flow.
Purpose of the Study:
- To investigate the role of membrane-bound APM in cerebral microvessels in metabolizing opioid peptides.
- To understand how APM activity in the brain's vasculature might influence opioid peptide levels and function.
Main Methods:
- Isolation and characterization of membrane-bound APM from hog and rabbit cerebral microvessels.
- Assays to measure the degradation of various opioid peptides by APM.
- Inhibition studies using specific peptidase inhibitors (amastatin, bestatin) and competitive inhibition assays with other neuropeptides.
Main Results:
- Cerebral microvessels from both hog and rabbit contain membrane-bound APM that degrades opioid peptides at neutral pH.
- Degradation primarily involves hydrolysis of the N-terminal Tyr-Gly bond, with shorter peptides like enkephalins being degraded more rapidly than beta-endorphin.
- Amastatin and bestatin potently inhibited APM activity, while other inhibitors were ineffective.
- Neurotensin and substance P competitively inhibited enkephalin degradation by microvascular APM.
Conclusions:
- Cerebral microvascular APM plays a significant role in metabolizing opioid peptides within the brain's vasculature.
- This enzyme may modulate local cerebral blood flow by influencing peptide signaling.
- APM activity could act as a barrier, limiting the passage of circulating enkephalins across the blood-brain barrier.